DNA expression systems based on alphaviruses

ABSTRACT

The disclosure describes recombinant alphavirus RNA molecules and expression of heterologous proteins therefrom in animal cells. Recombinant alphaviruses of the present invention, when made to express an antigenic protein, can be administered as vaccines.

[0001] The present invention is related to DNA expression systems based on alphaviruses, which systems can be used to transform animal cells for use in the production of desired products, such as proteins and vaccines, in high yields.

[0002] The rapid development of biotechnology is to a large extent due to the introduction of recombinant DNA technique, which has revolutionized cellbiological and medical research by opening new approaches to elucidate the molecular mechanisms of the cell. With the aid of the techniques of cDNA cloning, large numbers of interesting protein molecules are characterized each year. Therefore, a lot of research activity is today directed to elucidate the relationship between structure and function of these molecules. Eventually this knowledge will increase our possibilities to preserve healthiness and combat diseases in both humans and animals. Indeed, there is today a growing list of new “cloned” protein products that are already used as pharmaceuticals or diagnostics.

[0003] In the recombinant DNA approaches to study biological questions, DNA expression systems are crucial elements. Thus, efficient DNA expression systems, which are simple and safe to use, give high yields of the desired product and can be used in a variety of host cells, especially also in mammalian cells, are in great demand.

[0004] Many attempts have been made to develop DNA expression systems, which fulfill these requirements. Often, viruses have been used as a source of such systems. However, up to date none of the existing viral expression systems fulfill all these requirements in a satisfying way. For instance, the Baculovirus expression system for cDNA is extremely efficient but can be used only in insect cells (see Reference 1 of the list of cited references; for the sake of convenience, in the following the cited references are only identified by the number they have on said list). As many important molecules will have to be produced and processed in cells of mammalian origin in order for them to become active, this system cannot be used in such cases. Furthermore, the Baculovirus cDNA expression system is not practically convenient for analysis of the relationship between structure and function of a protein because this involves in general the analysis of whole series of mutant variants. Today it takes about 6-8 weeks to construct a single Baculo recombinant virus for phenotype analyses. This latter problem is also true for the rather efficient Vaccinia recombinant virus and other contemporary recombinant virus cDNA expression systems (2,3). The procedure to establish stably transformed cell lines is also a very laborious procedure, and in addition, often combined with very low levels of protein expression.

[0005] Hitherto, most attempts to develop viral DNA expression systems have been based on viruses having DNA genomes or retroviruses, the replicative intermediate of the latter being double stranded DNA.

[0006] Recently, however, also viruses comprising RNA genomes have been used to develop DNA expression systems.

[0007] In EP 0 194 809 RNA transformation vectors derived from (+) strand RNA viruses are disclosed which comprise capped viral RNA that has been modified by insertion of exogenous RNA into a region non-essential for replication of said virus RNA genome. These vectors are used for expression of the function of said exogenous RNA in cells transformed therewith. The RNA can be used in solution or packaged into capsids. Furthermore, this RNA can be used to generate new cells having new functions, i.e. protein expression. The invention of said reference is generally claimed as regards host cells, (+) strand RNA viruses and the like. Nevertheless, it is obvious from the experimental support provided therein that only plant cells have been transformed and in addition only Bromo Mosaic virus, a plant virus, has been used as transformation vector. Although it is stated in said reference that it is readily apparent to those skilled in the art to convert any RNA virus-cell system to a useful expression system for exogenous DNA using principals described in the reference, this has not been proven to be true in at least the case of animal cell RNA viruses. The reasons for this seem to be several. These include:

[0008] 1) Inefficiencies in transfecting animal cells with in vitro transcribed RNA;

[0009] 2) Inefficiency of apparently replication competent RNA transcripts to start RNA replication after commonly used transfection procedures;

[0010] 3) The inability to produce high titre stocks of recombinant virus that does not contain any helper virus;

[0011] 4) The inability to establish stable traits of transformed cells expressing the function of the exogenous RNA.

[0012] In Proc. Natl. Acad. Sci. USA, Vol 84, 1987, pp 4811-4815 a gene expression system based on a member of the Alphavirus genus, viz. Sindbis virus, is disclosed which is used to express the bacterial CAT (chloramphenicol acetyltransferase) gene in avian cells, such as chicken embryo fibroblasts.

[0013] Xiong et al., Science, Vol 243, 1989, 1188-1191 also disclose a gene expression system based on Sindbis virus. This system is said to be efficient in a broad range of animal cells. Expression of the bacterial CAT gene in insect, avian and mammalian cells inclusive of human cells is disclosed therein.

[0014] Even though it is known from prior art that one member of the Alphavirus genus, the Sindbis virus, can tolerate insertion and direct the expression of at least one foreign gene, the bacterial chloramfenicol acetyl transferase (CAT) gene, it is evident from the results described that both systems described above are both ineffective in terms of exogenous gene expression and also very cumbersome to use. Hence, neither system has found any usage in the field of DNA expression in animal cells today.

[0015] In the first example a cDNA copy of a defective interfering (DI) virus variant of Sindbis virus was used to carry the CAT gene. RNA was transcribed in vitro and used to transfect avian cells and some CAT protein production could be demonstrated after infecting cells with wild-type Sindbis virus. The latter virus provided the viral replicase for expression of the CAT construct. The inefficiency of this system depends on 1) low level of initial DI-CAT RNA transfection (0.05-0.5% of cells) and 2) inefficient usage of the DI-CAT RNA for protein translation because of unnatural and suboptimal protein intitation translation signals. This same system also results in packaging of some of the recombinant DI-CAT genomes into virus particles. However, this occurs simultaneously with a very large excess of wild-type Sindbis virus production. Therefore, the usage of this mixed virus stock for CAT expression will be much hampered by the fact that most of the replication and translation activity of the cells infected with such a stock will deal with the wild-type and not with recombinant gene expression.

[0016] Much of the same problems are inherent to the other Sindbis expression system described. In this an RNA replication competent Sindbis DNA vector is used to carry the CAT gene. RNA produced in vitro is shown to replicate in animal cells and CAT activity is found. However, as only a very low number of cells are transfected the overall CAT production remains low. Another possible explanation for this is that the Sindbis construct used is not optimal for replication. Wild-type Sindbis virus can be used to rescue the recombinant genome into particles together with an excess of wild-type genomes and this mixed stock can then be used to express a CAT protein via infection. However, this stock has the same problems as described above for the recombinant DI system. The latter paper shows also that if virus is amplified by several passages increased titres of the recombinant virus particles can be obtained. However, one should remember that the titre of the wild-type virus will increase correspondingly and the original problem of mostly wild-type virus production remains. There are also several potential problems when using several passages to produce a mixed virus stock. As there is no selected pressure for preservation of the recombinant genomes these might easily 1) undergo rearrangements and 2) become outnumbered by wild-type genomes as a consequence of less efficient replication and/or packaging properties.

[0017] Another important aspect of viral DNA expression vectors is use thereof to express antigens of unrelated pathogens and thus they can be used as vaccines against such pathogens.

[0018] Development of safe and effective vaccines against viral diseases has proven to be quite a difficult task. Although many existing vaccines have helped to combat the worldwide spread of many infectious diseases, there is still a large number of infectious agents against which effective vaccines are missing. The current procedures of preparing vaccines present several problems: (1) it is often difficult to prepare sufficiently large amounts of antigenic material; (2) In many cases there is the additional hazard that the vaccine preparation is not killed or sufficiently attenuated; (3) Effective vaccines are often hard to produce since there is a major difficulty in presenting the antigenic epitope in an immunologically active form; (4) In the case of many viruses, genetic variations in the antigenic components results in the evolution of new strains with new serological specificities, which again creates a need for the development of new vaccines.

[0019] Two types of viral DNA vectors have been developed in order to overcome many of these problems in vaccine production. These either provide recombinant viruses or provide chimaeric viruses. The recombinant viruses contain a wild-type virus package around a recombinant genome. These particles can be used to infect cells which then produce the antigenic protein from the recombinant genome. The chimaeric viruses also contain a recombinant genome but this specifies the production of an antigen, usually as part of a normal virus structural protein, which then will be packaged in progeny particles and e.g. exposed on the surface of the viral spike proteins. The major advantages of these kind of virus preparations for the purpose of being used as a vaccine are 1) that they can be produced in large scale and 2) that they provide antigen in a natural form to the immunological system of the organism. Cells, which have been infected with recombinant viruses, will synthesize the exogenous antigen product, process it into peptides that then present them to T cells in the normal way. In the case of the chimaeric virus there is, in addition, an exposition of the antigen in the context of the subunits of the virus particle itself. Therefore, the chimaeric virus is also-called an epitope carrier.

[0020] The major difficulty with these kind of vaccine preparations are, how to ensure a safe and limited replication of the particles in the host without side effects. So far, some success has been obtained with vaccinia virus as an example of the recombinant virus approach (69) and of polio virus as an example of a chimaeric particle (70-72). As both virus variants are based on commonly used vaccine strains one might argue that they could be useful vaccine candidates also as recombinant respectively chimaeric particles (69-72).

[0021] However, both virus vaccines are combined with the risk for side effects, even severe ones, and in addition these virus strains have already been used as vaccines in large parts of the population in many countries.

[0022] As is clear from the afore mentioned discussion there is much need to develop improved DNA expression systems both for an easy production of important proteins or polypeptides in high yields in various kinds of animal cells and for the production of recombinant viruses or chimaeric viruses to be used as safe and efficient vaccines against various pathogenes.

[0023] Thus, an object of the present invention is to provide an improved DNA expression system based on virus vectors which can be used both to produce proteins and polypeptides and as recombinant virus or chimaeric virus, which system offers many advantages over prior art.

[0024] To that end, according to the present invention there is provided an RNA molecule derived from an alphavirus RNA genome and capable of efficient infection of animal host cells, which RNA molecule comprises the complete alphavirus RNA genome regions, which are essential to replication of the said alphavirus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof.

[0025] Alphavirus is a genus belonging to the family Togaviridae having single stranded RNA genomes of positive polarity enclosed in a nucleocapsid surrounded by an evelope containing viral spike proteins.

[0026] The Alphavirus genus comprises among others the Sindbis virus, the Semliki Forest virus (SFV) and the Ross River virus, which are all closely related.

[0027] According to a preferred embodiment of the invention, the Semliki Forest virus (SFV) is used as the basis of the DNA expression system.

[0028] The exogenous RNA sequence encodes a desired genetic trait, which is to be conferred on the virus or the host cell, and said sequence is usually complementary to a DNA or cDNA sequence encoding said genetic trait. Said DNA sequence may be comprised of an isolated natural gene, such as a bacterial or mammalian gene, or may constitute a synthetic DNA sequence coding for the desired genetic trait i.e. expression of a desired product, such as an enzyme, hormone, etc. or expression of a peptide sequence defining an exogenous antigenic epitope or determinant.

[0029] If the exogenous RNA sequence codes for a product, such as a protein or polypeptide, it is inserted into the viral RNA genome replacing deleted structural protein encoding region(s) thereof, whereas a viral epitope encoding RNA sequence may be inserted into structural protein encoding regions of the viral RNA genome, which essentially do not comprise deletions or only have a few nucleosides deleted.

[0030] The RNA molecule can be. used per se, e.g. in solution to transform animal cells by conventional transfection, e.g. the DEAE-Dextran method or the calcium phosphate precipitation method. However, the rate of transformation of cells, and, thus the expression rate can be expected to increase substantially if the cells are transformed by infection with infectious viral particles. Thus, a suitable embodiment of the invention is related to an RNA virus expression vector comprising the RNA molecule of this invention packaged into infectious particles comprising the said RNA within the alphavirus nucleocapsid and surrounded by the membrane including the alphavirus spike proteins.

[0031] The RNA molecule of the present invention can be packaged into such particles without restraints provided that it has a total size corresponding to the wild type alphavirus RNA genome or deviating therefrom to an extent compatible with package of the said RNA into the said infectious particles.

[0032] These infectious particles, which include recombinant genomes packaged to produce a pure, high titre recombinant virus stock, provides a means for exogenous genes or DNA sequences to be expressed by normal virus particle infection, which as regards transformation degree, is much more efficient than RNA transfection.

[0033] According to a suitable embodiment of the invention such infectious particles are produced by cotransfection of animal host cells with the present RNA which lacks part of or the complete region(s) encoding the structural viral proteins together with a helper RNA molecule transcribed in vitro from a helper DNA vector comprising the SP6 promoter region, those 5′ and 3′ regions of the alphavirus cDNA which encode cis acting signals needed for RNA replication and the region encoding the viral structural proteins but lacking essentially all of the nonstructural virus proteins encoding regions including sequenses encoding RNA signals for packaging of RNA into nucleocapsid particles, and culturing the host cells.

[0034] According to another aspect of the invention efficient introduction of the present RNA into animal host cells can be achieved by electroporation. For example, in the case of Baby Hamster Kidney (BHK) cells a transformation degree of almost 100% has been obtained for the introduction of an RNA transcript derived from SFV cDNA of the present invention. This rakes it possible to reach so high levels of exogenous protein production in every cell that the proteins can be followed in total cell lysates without the need of prior concentration by antibody precipitation.

[0035] By electroporation, it is also possible to obtain a high degree of cotransfection in the above process for production of infectious particles comprising packaged RNA of the present invention. Essentially all animal cells will contain both the present RNA molecule and the helper RNA molecule, which leads to a very efficient trans complementation and formation of infectious particles. A pure recombinant virus stock, consisting of up to 10⁹-10¹⁰ infectious particles, can be obtained from 5×10⁶ cotransfected cells after only a 24 h incubation. Furthermore, the so obtained virus stock is very safe to use, since it is comprised of viruses containing only the desired recombinant genome, which can infect host cells but can not produce new progeny virus.

[0036] Theoretically, a regeneration of a wild-type virus genome could take place when producing the recombinant virus in the contransfected cells. However, the possibility to avoid spread of such virus can be eliminated by incorporating a conditionally lethal mutation into the structural part of the helper genome. Such a mutation is described in the experimental part of this application. Thus, the virus produced with such a helper will be noninfectious if not treated in vitro under special conditions.

[0037] The technique of electroporation is well known within the field of biotechnology and optimal conditions can be established by the man skilled in the art. For instance, a BioRad Gene pulser apparatus (BioRad, Richmond, Calif., USA) can be used to perform said process.

[0038] The RNA molecule of the present invention is derived by in vivo or in vitro transcription of a cDNA clone, originally produced from an alphavirus RNA and comprising an inserted exogenous DNA fragment encoding a desired genetic trait.

[0039] Accordingly, the present invention is also related to a DNA expression vector comprising a full-length or partial cDNA complementary to alphavirus RNA or parts thereof and located immediately downstream of the SP6 RNA polymerase promoter and having a 5′ATGG, a 5′GATGG or any other 5′ terminus and a TTTCCA₆₉ACTAGT or any other 3′ terminus.

[0040] According to one aspect of the present invention portions of the viral cDNA are deleted, the deletions comprising the complete or part of the region(s) encoding the virus structural proteins, and the vector further comprises an integrated polylinker region, which may correspond to BamHI-SmaI-XmaI, inserted at a location which enables an exogenous DNA fragment encoding a foreign polypeptide or protein to be inserted into the vector cDNA for subsequent expression in an animal host cell.

[0041] According to another aspect of this invention, the vector is comprised of full-length cDNA wherein an exogenous DNA fragment encoding a foreign epitopic peptide sequence can be inserted into a region coding for the viral structural proteins.

[0042] It is appreciated that this cDNA clone with its exogenous DNA insert is very efficiently replicated after having been introduced into animal cells by transfection.

[0043] A very important aspect of the present invention is that it is applicable to a broad range of host cells of animal origin. These host cells can be selected from avian, mammalian, reptilian, amphibian, insect and fish cells. Illustrative of mammalian cells are human, monkey, hamster, mouse and porcine cells. Suitable avian cells are chicken cells, and as reptilian cells viper cells can be used. Cells from frogs and from mosquitoes and flies (Drosophila) are illustrative of amphibian and insecticidal cells, respectively. A very efficient virus vector/host cell system according to the invention is based on SFV/BHK cells, which will be discussed more in detail further below.

[0044] However, even though a very important advantage of the present DNA expression vector is that it is very efficient in a broad variety of animal cells it can also be used in other eucaryotic cells and in procaryotic cells.

[0045] The present invention is also related to a method to produce transformed animal host cells comprising transfection of the cells with the present RNA molecule or with the present transcription vector comprised of cDNA and carrying an exogenous DNA fragment. According to a suitable embodiment of the invention, transfection is produced by the above mentioned electroporation method, a very high transfection rate being obtained.

[0046] A further suitable transformation process is based on infection of the animal host cells with the above mentioned infectious viral particles comprising the present RNA molecule.

[0047] The transformed cells of the present invention can be used for different purposes.

[0048] One important aspect of the invention is related to use of the present transformed cells to produce a polypeptide or a protein by culturing the transformed cells to express the exogenous RNA and subsequent isolation and purification of the product formed by said exepression. The transformed cells can be produced by infection with the present viral particles comprising exogenous RNA encoding the polypeptide or protein as mentioned above, or by transfection with an RNA transcript obtained by in vitro transcription of the present DNA vector comprised of cDNA and carrying an exogenous DNA fragment coding for the polypeptide or the protein.

[0049] Another important aspect of the invention is related to use of the present transformed cells for the production of antigens comprised of chimaeric virus particles for use as immunizing component in vaccines or for immunization purposes for in vivo production of immunizing components for antisera production.

[0050] Accordingly, the present invention is also related to an antigen consisting of a chimaeric alphavirus having an exogenous epitopic peptide sequence inserted into its structural proteins.

[0051] Preferably, the chimaeric alphavirus is derived from SFV.

[0052] According to a suitable embodiment, the exogenous epitopic peptide sequence is comprised of an epitopic peptide sequence derived from a structural protein of a virus belonging to the immunodeficiency virus class inclusive of the human immunodeficiency virus types.

[0053] A further aspect of the invention is related to a vaccine preparation comprising the said antigen as immunizing component.

[0054] In said vaccine the chimaeric alphavirus is suitably attenuated by comprising mutations, such as the conditionally lethal SFV-mutation described before, amber (stop codon) or temperature sensitive mutations, in its genome.

[0055] For instance, if the chimaeric virus particles containing the afore mentioned conditional lethal mutation in its structural proteins (a defect to undergo a certain proteolytical cleavage in host cell during morphogenesis) is used as a vaccine then this is first activated by limited proteolytic treatment before given to the organism so that it can infect recipient cells. New chimaeric particles will be formed in cells infected with the activated virus but these will again be of the lethal phenotype and further spread of infection is not possible.

[0056] The invention is also concerned with a method for the production of the present antigen comprising

[0057] a) in vitro transcription of the cDNA of the present DNA vector carrying an exogenous DNA fragment encoding the foreign epitopic peptide sequence and transfection of animal host cells with the produced RNA transcript, or

[0058] b) transfection of animal host cells with the said cDNA of the above step a), culturing the transfected cells and recovering the chimaeric alphavirus antigen. Preferably, transfection is produced by electroporation.

[0059] Still another aspect of the invention is to use a recombinant virus containing exogenous RNA encoding a polypeptide antigen for vaccination purpose or to produce antisera. In this case the recombinant virus or the conditionally lethal variant of it is used to infect cells in vivo and antigen production will take place in the infectious cells and used for antigen presentation to the immunological system.

[0060] According to another embodiment of the invention, the present antigen is produced in an organism by using in vivo infection with the present infectious particles containing exogenous RNA encoding an exogenous epitopic peptide sequence.

[0061] In the following, the present invention will be illustrated more in detail with reference to the Semliki Forest virus (SFV), which is representative for the alphaviruses. This description can be more fully understood in conjunction with the accompanying drawings in which:

[0062]FIG. 1 is a schematic view over the main assembly and disassembly events involved in the life cycle of the Semliki Forest virus, and also shows regulation of the activation of SFV entry functions by p62 cleavage and pH;

[0063]FIG. 2 illustrates the use of translocation signals during synthesis of the structural proteins of SFV; top, the gene map of the 26S subgenomic RNA; middle, the process of membrane translocation of the p62, 6K and E1 proteins; small arrows on the lumenal side denote signal peptidase cleavages; at the bottom, the characteristics of the three signal peptides are listed;

[0064]FIG. 3 shows features that make SFV an excellent choice as an expression vector;

[0065] FIGS. 4A-C show the construction of full-length infectious clones of SFV;

[0066]FIG. 4A shows a schematic restriction map of the SFV genome; primers used for initiating cDNA synthesis are indicated as arrows, and the cDNA inserts used to assemble the final clone are showed as bars;

[0067]FIG. 4B shows plasmid pPLH211, i.e. the SP6 expression vector used as carrier for the full-length infectious clone of SFV, and the resulting plasmid pSP6-SFV4;

[0068]FIG. 4C shows the structure of the SP6 promoter area of the SFV clone; the stippled bars indicate the SP6 promoter sequence, and the first necleotide to be transcribed is marked by an asterisk; underlined regions denote authentic SFV sequences;

[0069]FIG. 5 shows the complete nucleotide sequence of the pSP6-SFV4 RNA transcript as DNA (U=T) and underneath the DNA sequence, the amino acid sequence of the non-structural polyprotein and the structural polyprotein;

[0070]FIG. 6 shows an SFV cDNA expression system for the production of virus after transfection of in vitro made RNA into cells;

[0071]FIG. 7 shows the construction of the SFV expression vectors pSFV1-3 and of the Helper 1;

[0072]FIG. 8 shows the polylinker region of SFV vector plasmids pSFV1-3; the position of the promoter for the subgenomic 26S RNA is boxed, and the first nucleotide to be transcribed is indicated by an asterisk;

[0073]FIG. 9 is a schematic presentation of in vivo packaging of pSFV1-dhfr RNA into infectious particles using helper trans complementation; (dhfr means dihydrofolate reductase)

[0074]FIG. 10 shows the use of trypsin to convert p62-containing noninfectious virus particles to infectious particles by cleavage of p62 to E2 and E3;

[0075]FIG. 11 shows the expression of heterologous proteins in BHK cells upon RNA transfection by electroporation; and

[0076]FIG. 12 shows in its upper part sequences encompassing the major antigenic site of SFV and the in vitro made substitutions leading to a BamHI restriction endonuclease site, sequences spanning the principal neutralizing domain of the HIV gp120 protein, and the HIV domain inserted into the SFV carrier protein E2 as a BamHI oligonucleotide; and its lower part is a schematic presentation of the SFV spike structure with blow-ups of domain 246-251 in either wild type or chimaeric form.

[0077] The alphavirus Semliki Forest virus (abbreviated SFV in the following text) has for some 20 years been used as model system in both virology and cell biology to study membrane biosynthesis, membrane structure and membrane function as well as protein-RNA interactions (4, 5). The major reason for the use of SFV as such a model is due to its simple structure and efficient replication.

[0078] With reference to FIGS. 1-3, in the following the SFV and its replication are explained more in detail. In essential parts, this disclosure is true also for the other alphaviruses, such as the Sindbis virus, and many of the references cited in this connection are indeed directed to the Sindbis virus. SFV consists of an RNA-containing nucleocapsid and a surrounding membrane composed of a lipid bilayer and proteins, a regularly arranged icosahedral shell of a protein called C protein forming the capsid inside which the genomic RNA is packaged. The capsid is surrounded by the lipid bilayer that contains three proteins called E1, E2, and E3. These so-called envelope proteins are glycoproteins and their glycosylated portions are on the outside of the lipid bilayer, complexes of these proteins forming the “spikes” that can be seen in electron micrographs to project outward from the surface of the virus.

[0079] The SFV genome is a single-stranded 5′-capped and 3′-polyadenylated RNA molecule of 11422 nucleotides (6,7).

[0080] It has positive polarity, i.e. it functions as an mRNA, and naked RNA is able to start an infection when introduced into the cytoplasm of a cell. Infection is initiated when the virus binds to protein receptors on the host cell plasma membrane, whereby the virions become selectively incorporated-into “coated pits” on the surface of the plasma membrane, which invaginate to form coated vesicles inside the cell, whereafter said vesicles bearing endocytosed virions rapidly fuse with organelles called endosomes. From the endosome, the virus escapes into the cell cytosol as the bare nucleocapsid, the viral envelope remaining in the endosome. Thereafter, the nucleocapsid is “uncoated” and, thus, the genomic RNA is released. Referring now to FIG. 1, infection then proceeds with the translation of the 5′ two-thirds of the genome into a polyprotein which by self-cleavage is processed to the four nonstructural proteins nsP1-4 (8). Protein nsP1 encodes a methyl transferase which is responsible for virus-specific capping activity as well as initiation of minus strand synthesis (9, 10); nsP2 is the protease that cleaves the polyprotein into its four subcomponents (11, 12); nsP3 is a phosphoprotein (13, 14) of as yet unknown function, and nsP4 contains the SFV RNA polymerase activity (15, 16). Once the nsP proteins have been synthesized they are responsible for the replication of the plus strand (42S) genome into full-length minus strands. These molecules then serve as templates for the production of new 42S genomic RNAs. They also serve as templates for the synthesis of subgenomic (26S) RNA. This 4073 nucleotides long RNA is colinear with the last one-third of the genome, and its synthesis is internally initiated at the 26S promoter on the 42S minus strands (17, 18).

[0081] The capsid and envelope proteins are synthesized in different compartments, and they follow separate pathways through the cytoplasm, viz. the envelope proteins are synthesized by membrane-bound ribosomes attached to the rough endoplasmic reticulum, and the capsid protein is synthesized by free ribosomes in the cytosol. However, the 26S RNA codes for all the structural proteins of the virus, and these are synthesized as a poly-protein precursor in the order C-E3-E2-6K-E1 (19). Once the capsid (C) protein has been synthesized it folds to act as a protease cleaving itself off the nascent chain (20, 21). The synthesized C proteins bind to the recently replicated genomic RNA to form new nucleocapsid structures in the cell cytoplasm.

[0082] The said cleavage reveals an N-terminal signal sequence in the nascent chain which is recognized by the signal recognition particle targeting the nascent chain-ribosome complex to the endoplasmic reticulum (ER) membrane (22, 23), where it is cotranslationally translocated and cleaved by signal peptidase to the three structural membrane proteins p62 (precursor form of E3/E2), 6K and E1 (24, 25). The translocational signals used during the synthesis of the structural proteins are illustrated in FIG. 2. The membrane proteins undergo extensive posttranslational modifications within the biosynthetic transport pathway of the cell. The p62 protein forms a heterodimer with E1 via its E3 domain in the endoplasmic reticulum (26). This dimer is transported out to the plasma membrane, where virus budding occurs through spike nucleocapsid interactions. At a very late (post-Golgi) stage of transport the p62 protein is cleaved to E3 and E2 (27), the forms that are found in mature virions. This cleavage activates the host cell binding function of the virion as well as the membrane fusion potential of E1. The latter activity is expressed by a second, low-pH activation step after the virus enters the endosomes of a new host cell and is responsible for the release of the viral nucleocapsid into the cell cytoplasm (28-32). The mature virus particles contain one single copy of the RNA genome encapsidated within 180 copies of the capsid protein in a T=3 symmetry, and is surrounded by a lipid bilayer carrying 240 copies of the spike trimer protein consisting of E1+E2+E3 arranged in groups of three in a T=4 symmetry (33).

[0083] The SFV entry functions are activated and regulated by p62 cleavage and pH. More specifically, the p62-E1 heterodimers formed in the ER are acid resistant. When these heterodimers are transported to the plasma membrane via the Golgi complex the E1 fusogen cannot be activated in spite of the mildly acidic environment, since activation requires dissociation of the complex. As is illustrated in FIG. 1, the released virus particles contain E2E1 complexes. Since the association between E2 and E1 is sensitive to acidic pH, during entry of the virus into a host cell through endocytosis the acidic milieu of the endosome triggers the dissociation of the spike complex (E1E2 E3) resulting in free E1. The latter can be activated for the catalysis of the fusion process between the viral and endosomal membranes in the infection process as disclosed above.

[0084] As indicated in the preceding parts of the disclosure, the alphavirus system, and especially the SFV system, has several unique features which are to advantage in DNA expression systems. These are summarized below with reference to FIG. 3.

[0085] 1. Genome of positive polarity. The SFV RNA genome is of positive polarity, i.e. it functions directly as mRNA, and infectious RNA molecules can thus be obtained by transcription from a full-length cDNA copy of the genome.

[0086] 2. Efficient replication. The infecting RNA molecule codes for its own RNA replicase, which in turn drives an efficient RNA replication. Indeed, SFV is one of the most efficiently replicating viruses known. Within a few hours up to 200.000 copies of the plus-RNAs are made in a single cell. Because of the abundance of these molecules practically all ribosomes of the infected cell will be enrolled in the synthesis of the virus encoded proteins, thus overtaking host protein synthesis (34), and pulse-labelling of infected cells results in almost exclusive labelling of viral proteins. During a normal infection 10⁵ new virus particles are produced from one single cell, which calculates to at least 10⁸ protein molecules encoded by the viral genome (5).

[0087] 3. Cytoplasmic replication. SFV replication occurs in the cell cytoplasm, where the virus replicase transcribes and caps the subgenomes for production of the structural proteins (19). It would obviously be very valuable to include this feature in a cDNA expression system to eliminate the many problems that are encountered in the conventional “nuclear” DNA expression systems, such as mRNA splicing, limitations in transcription factors, problems with capping efficiency and mRNA transport.

[0088] 4. Late onset of cytopathic effects. The cytopathic effects in the infected cells appear rather late during infection. Thus, there is an extensive time window from about 4 hours after infection to up to 24 hours after infection during which a very high expression level of the structural proteins is combined with negligible morphological change.

[0089] 5. Broad host range. This phenomenon is probably a consequence of the normal life cycle which includes transmission through arthropod vectors-to wild rodents and birds in nature. Under laboratory conditions, SFV infects cultured mammalian, avian, reptilian and insect cells (35) (Xiong, et al, loc. cit.)

[0090] 6. In nature SFV is of very low pathogenicity for humans. In addition, the stock virus produced in tissue culture cells is apparently apathogenic. By means of specific mutations it is possible to create conditionally lethal mutations of SFV, a feature that is of great use to uphold safety when massproduction of virus stocks is necessary.

[0091] In the nucleotide and amino acid sequences the following abbreviations have been used in this specification:

[0092] Ala, alanine; lle, isoleucine; leu, leucine; Met, methionine; Phe, phenylalanine; Pro, proline; Trp, tryptophan; Val, valine; Asn, asparagine; Cys, cysteine; Gln, glutamine; Gly, glycine; Ser, serine; Thr, threonine; Tys, tyrosine; Arg, arginine; His, histidine; Lys, lysine; Asp, aspartic acid; Glu, glutamic acid; A, adenine; C, cytosine; G, guanine; T, thymine; U, uracil.

[0093] The materials and the general methodology used in the following examples are disclosed below.

[0094] 1. Materials. Most restriction enzymes, DNA Polymerase I, Klenow fragment, calf intestinal phosphatase, T4 DNA ligase and T4 Polynucleotide kinase were from Boehringer (Mannheim, FRG). SphI, StuI and KpnI together with RNase inhibitor (RNasin) and SP6 Polymerase were from Promega Biotec (Madison, Wis.). Sequenase (Modified T7 polymerase) was from United States Biochemical (Cleveland, Ohio). Proteinase K was from Merck (Darmstadt, FRG). Ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides and the cap analogue m⁷G(5′)ppp(5′)G were from Pharmacia (Sweden). Oligonucleotides were produced using an Applied Bio-systems synthesizer 380B followed by HPLC and NAP-5 (Pharmacia) purification. Spermidine, phenylmethylsulfonyl fluoride (PMSF), diethylpyrocarbonate (DEPC), bovine serum albumin (BSA), creatine phosphate and creatine phosphokinase were from Sigma (St. Louis, Mo.). Pansorbin was from CalBiochem (La Jolla, Calif.). Agarose was purchased from FMC BioProducts (Rockland, Me.), and acrylamide from BioRad (Richmond, Calif.). L-[³⁵S]-methionine and α-[³⁵S]-dATP-α-S were from Amersham.

[0095] 2. Virus growth and purification: BHK-21 cells were grown in BHK medium (Gibco Life Technologies, Inc., N.Y.) supplemented with 5% fetal calf serum, 10% tryptose phosphate broth, 10 mM HEPES (N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid) and 2 mM glutamine. 90% confluent monolayers were washed once with PBS and infected with SFV in MEM containing 0.2% bovine serum albumin (BSA), 10 mM HEPES and 2 mM glutamine at a multiplicity of 0.1. Twenty-four hours post infection (p.i.) the medium was collected and cell debris removed by centrifugation at 8,000× g for 20 min at 4° C. The virus was pelleted from the medium by centrifugation at 26,000 rpm for 1.5 h in an SW28 rotor at 4° C. The virus was resuspended in TN containing 0.5 mM EDTA.

[0096] 3. Metabolic labeling and immunoprecipitation. Confluent monolayers of BHK cells grown in MEM supplemented with 10 mM HEPES, 2 mM glutamine, 0.2% BSA, 100 IU/mol of penicillin and 100 μg/ml streptomycin, were infected at a multiplicity of 50 at 37° C. After 1 h p.i. the medium was replaced with fresh and growth continued for 3.5 h. The medium was removed and cells washed once with PBS and overlayed with methionine-free MEM containing 10 mM HEPES and 2 mM glutamine. After 30 min at 37° C. the medium was replaced with the same containing 100 μCi/ml of [³⁵S]methionine (Amersham) and the plates incubated for 10 min at 37° C. The cells were washed twice with labeling medium containing 10× excess methionine and then incubated in same medium for various times. The plates were put on ice, cells washed once with ice-cold PBS and finally lysis buffer (1% NP-40-50 mM Tris-HCl, pH 7.6-150 mM NaCl-2 mM EDTA) containing 10 μg/ml PMSF (phenylmethylsulfonyl fluoride) was added. Cells were scraped off the plates, and nuclei removed by centrifugation at 6,000 rpm for 5 min at 4° C. in an Eppendorf centrifuge. Immunoprecipitations of proteins was performed as described (31). Briefly, antibody was added to lysate and the mixture kept on ice for 30 min. Complexes were recovered by binding to Pansorbin for 30 min on ice. Complexes were washed once with low salt buffer, once with high salt buffer, and once with 10 mM Tris-HCl, pH 7.5, before heating with gel loading buffer. To precipitate dhfr, SDS was added to 0.1% and the mixture heated to 95° C. for 2 min followed by addition of 10 volumes of lysis buffer. Anti-E1 [8.139], anti-E2 [5.1] (36), and anti-C [12/2] (37) monoclonals have been described. The human transferrin receptor was precipitated with the monoclonal antibody OKT-9 in ascites fluid. This preparation was provided by Thomas Ebel at our laboratory using a corresponding hybridoma cell line obtained from ATCC (American Typ Culture Collection) No CRL 8021. Polyclonal rabbit anti-mouse dhfr was a kind gift from E. Hurt (European Molecular Biology Laboratory, Heidelberg, FRG) and rabbit anti-lysozyme has been described

[0097] 4. Imniunofluorescence. To perform indirect immunofluorescence, infected cell monolayers on glass cover-slips were rinsed twice with phosphate-buffered saline (PBS) and fixed in -20° C. methanol for 6 min. After fixation, the methanol was removed and the coverslip washed 3 times with PBS. Unspecific antibody binding was blocked by incubation at room temperature with PBS containing 0.5% gelatin and 0.25% BSA. The blocking buffer was removed and replaced with same buffer containing primary antibody. After 30 min at room temperature the reaction was stopped by washing 3 times with PBS. Binding of secondary antibody (FITC-conjugated sheep anti-mouse [BioSys, Compiegne, France]) was done as for the primary antibody. After 3 washes with PBS and one rinse with water the coverslip was allowed to dry before mounting in Moviol 4-88 (Hoechst, Frankfurt am Main, FRG) containing 2.5% DABCO (1,4-diazobicyclo [2.2.2]-octane).

[0098] 5. DNA procedures. Plasmids were grown in Escherichia coli DH5α (Bethesda Research Laboratories) [recA endAl gyrA96 thi1 hsdR17 supE44 relA1 A(lacZYA-argF)U169φ80dlacZΔ(M15)]. All basic DNA procedures were done essentially as described (39). DNA fragments were isolated from agarose gels by the freeze-thaw method (40) including 3 volumes of phenol during the freezing step to increase yield and purity. Fragments were purified by benzoyl-naphthoyl-DEAE (BND) cellulose (Serva Feinbiochemica, Heidelberg, FRG) chromatography (41). Plasmids used for production of infectious RNA were purified by sedimentation through 1 M NaCl followed by banding in CsCl (39). In some cases plasmids were purified by Qiagen chromatography (Diagen Gmbh, Dusseldorf, FRG).

[0099] 6. Site-directed oligonucleotide mutagenesis. For oligonucleotide mutagenesis, relevant fragments of the SFV cDNA clone were subcloned into M13 mp18 or mp 19 (42) and transformed (43) into DH5αFIQ [enda1 hsdR1 supE44 thil recA1 gyrA96 relA1 φ80dlacΔ (M15) Δ(lacZYA-argF)U169/F′proAB lacl^(q) lacZΔ(M15) Tn 5] (Bethesda Research Laboratories). RF DNA from these constructs was transformed into RZ1032 (44) [Hfr KL16 dut1 ung1 thil relA1 supE44 zbd279:Tn10.], and virus grown in the presence of uridine to incorporate uracil residues into the viral genome. Single stranded DNA was isolated by phenol extraction from PEG precipitated phage. Oligonucleotides were synthesized on an Applied Biosystems 380B synthesizer and purified by gel filtration over NAP-5 columns (Pharmacia). The oligonucleotides 5′-CGGCCAGTGAATTCTGATTGGATCCCGGGTAATTAATTGAATTACATCCCTACGCAAACG, 5′-GCGCACTATTATAGCACCGGCTCCCGGGTAATTAATTGACGCAAACGTTTTACGGCCGCCGG and 5′-GCGCACTATTATAGCACCATGGATCCGGGTAATTAATTGACGTTTTACGGCCGCCGGTGGCG were used to insert the new linker sites [BamHI-SmaI-XmaI] into the SFV cDNA clone. The oligonucleotides 5′-CGGCGGTCCTAGATTGGTGCG and 5′-CGCGGGCGCCACCGGCGGCCG were used as sequencing primers (SP1 and SP2) up- and downstream of the polylinker site. Phosphorylated oligonucleotides were used in mutagenesis with Sequenase (Unites States Biochemicals, Cleveland, Ohio) as described earlier (44, 45). In vitro made RF forms were transformed into DH5αF′IQ and the resulting phage isolates analyzed for the presence of correct mutations by dideoxy sequencing according to the USB protocol for using Sequenase. Finally, mutant fragments were reinserted into the full-length SFV cDNA clone. Again, the presence of the appropriate mutations was verified by sequencing from the plasmid DNA. Deletion of the 6K region has been described elsewhere.

[0100] 7. In vitro transcription. SpeI linearized plasmid DNA was used as template for in vitro transcription. 15 RNA was synthesized at 37° C. for 1 h in 10-50 μl reactions containing 40 mM Tris-HCl (pH 7.6), 6 mM spermidine-HCl, 5 mM dithiothreitol (DTT), 100 μg/ml of nuclease free BSA, 1 mM each of ATP, CTP and UTP, 500 μM of GTP, 1 unit/μl of RNasin and 100-500 units/ml of SP6 RNA polymerase. For production of capped transcripts (46), the analogs m G(5′)ppp(5′)G or m⁷G(5′)ppp(5′)A were included in the reaction at 1 mM. For quantitation of RNA production, trace amounts of [α-³²P]-UTP (Amersham) was included in the reactions and incorporation measured from trichloroacetic acid precipitates. When required, DNA or RNA was digested at 37° C. for 10 min by adding DNase 1 or RNase A at 10 units/μg template or 20 μg/ml respectively.

[0101] 8. RNA transfection. Transfection of BHK monolayer cells by the DEAE-Dextran method was done as described previously (47). For transfection by electroporation, RNA was added either directly from the in vitro transcription reaction or diluted with transcription buffer containing 5 mM DTT and 1 unit/μl of RNasin. Cells were trypsinized, washed once with complete BHK-cell medium and once with ice-cold PBS (without MgCl₂ and CaCl₂) and finally resuspended in PBS to give 10⁷ cells/ml. Cells were either used directly or stored (in BHK medium) on ice over night. For electroporation, 0.5 ml of cells were transferred to a 0.2 cm cuvette (BioRad), 10-50 μl of RNA solution added and the solution mixed by inverting the cuvette. Electroporation was performed at room temperature by two consecutive pulses at 1.5 kV/25 pF using a BioRad Gene Pulser apparatus with its pulse controller unit set at maximum resistance. After incubation for 10 min, the cells were diluted 1:20 in complete BHK-cell medium and transferred onto tissue culture plates. For plaque assays, the electroporated cells were plated together with about 3×10⁵ fresh cells per ml and incubated at 37° C. for 2 h, then overlayed with 1.8% low melting point agarose in complete BHK-cell medium. After incubation at 37° C. for 48 h, plaques were visualized by staining with neutral red.

[0102] 9. Gel electrophoresis. Samples for sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) were prepared and run on 12% separating gels with a 5% stacking gel as previously described (48). For re-solving the 6K peptide, a 10% -20% linear acrylamide gradient gel was used. Gels were fixed in 10% acetic acid -30% methanol for 30 min before exposing to Kodak XAR-5 film. When a gel was prepared for fluorography (49), it was washed after fixation for 30 min in 30% methanol and then soaked in 1M sodium salicylate—30% methanol for 30 min before drying. Nucleic acids were run on agarose gels using 50 mM Tris-borate—2.5 mM Na₂EDTA as buffer. For staining 0.2 μg/ml of ethidium bromide was included in the buffer and gel during the run.

EXAMPLE 1

[0103] In this example a full-length SFV cDNA clone is prepared and placed in a plasmid containing the SP6 RNA polymerase promoter to allow in vitro trancription of full-length and infectious transcripts. This plasmid which is designated pSP6-SFV4 has been deposited on 28 NOV 1991 at PHLS Centre for Applied Microbiology & Research

[0104] European Collection of Animal Cell Cultures, Porton Down, Salisbury, Wiltshire, U.K:, and given the provisional accession number 91112826.

[0105] As illustrated in FIG. 4A-C the strategy for construction the SFV clone was to prime cDNA synthesis on several positions along the template RNA downstream of suitable restriction endonuclease sites defined by the known nucleotide sequence of the SFV RNA molecule. Virus RNA was isolated by phenol-chloroform extraction from purified virus (obtainable among others from the Arbovirus collection in Yale University, New Haven, USA) and used as template for cDNA synthesis as previously described (50). First strand synthesis was primed at three positions, using 5′-TTTCTCGTAGTTCTCCTCGTC as primer-1 (SFV coordinate 2042-2062) and 5′-GTTATCCCAGTGGTTGTTCTCGTAATA as primer-2 (SFV coordinate 3323-3349) and an oligo-dT₁₂₋₁₈ as primer −3 (3′ end of SFV) FIG. 4A).

[0106] Second strand synthesis was preceded by hybridization of the oligonucleotide 5′-ATGGCGGATGTGTGACATACACGACGCC (identical to the 28 first bases of the genome sequence of SFV) to the first strand cDNA. After completion of second strand synthesis cDNA was trimmed and in all cases except in the case of the primer-1 reaction, the double-stranded adaptor 5′-AATTCAAGCTTGCGGCCGCACTAGT/GTTCGAACGCCGGCGTGATCA-3′ (5′-sticky-EcoRI-HindIII-NotI-XmaIII-SpeI-blunt-3′) was added and the cDNa cloned into EcoR1 cleaved pTZ18R (Pharmacia, Sweden) as described (51). The cloning of the 5′ end region was done in a different way. Since SFV contains a HindIII site at position 1947, cDNA primed with primer-1 should contain this area and therefore HindIII could be used to define the 3′ end of that cDNA. To obtain a restriction site at the very 5′ end of the SFV, cDNA was cloned into SmaI-HindIII cut pGEM1 (Promega Biotec., Madison, Wis.). Since the SFV genome starts with the sequence 5′-ATGG, ligation of this onto the blunt CCC-3′ end of the SmaI site created an NcoI site C′CATGG. Although the SFV sequence contains 3 NcoI sites, none of these are within the region preceding the HindiII site, and thus these 5′ end clones could be further subcloned as NcoI-HindIII fragments into a vector especially designed for this purpose (see below). The original cDNA clones in pGEM1 were screened by restriction analysis and all containing inserts bigger than 1500 bp were selected for further characterization by sequencing directly from the plasmid into both ends of the insert, using SP6 or T7 sequencing primers. The SFV 5′-end clones in pTZ18R were sequenced using lac sequencing primers. To drive in vitro synthesis of SFV RNA the SP6 promoter was used. Cloning of the SFV 5′ end in front of this promoter without adding too many foreign nucleotides required that a derivative of pGEM1 had to be constructed. Hence, pGEM1 was opened at EcoR1 and Bal31 deletions were created, the DNA blunted with T4 DNA polymerase and an Ncol oligonucleotide (5′-GCCATGGC) added. The clones obtained were screened by colony hybridization (39). with the oligonucleotide 5′-GGTGACACTATAGCCATGGC designed to pick up (at suitable stringency) the variants that had the NcoI sequence immediately at the transcription initiation site of the SP6 promoter (G underlined). Since the Bal3l deletion had removed all restriction sites of the multicloning site of the original plasmid, these were restored by cloning a PvuI-NcoI fragment from the new variant into another variant of pGEM1 (pDH101) that had an NcoI site inserted at its HindIII position in the polylinker. This created the plasmid pDH201. Finally, the adaptor used for cloning the SFV cDNA was inserted into pDH201 between the EcoRI and PvuII sites to create plasmid pPLH211 (FIG. 4B). This plasmid was then used as recipient for SFV cDNA fragments in the assembly of the full-length clone by combining independent overlapping subclones using these sites. The fragments and the relevant restriction sites used to assemble the full-length clone, pSP6-SFV4, are depicted in (FIG. 4A). For the 5′-end, the selected fragment contained the proper SFV sequence 5′-ATGG, with one additional G-residue in front. When this G-residue was removed it reduced transcription efficiency from SP6 but did not affect infectivity of the in vitro made RNA. Thus, the clone used for all subsequent work contains the G-residue at the 5′ end. For the 3′-end of the clone, a cDNA fragment containing 69 A-residues was selected. By inclusion of the unique SpeI site at the 3′-end of the cDNa, the plasmid can be linearized to allow for runoff transcription in vitro giving RNA-carrying 70 A residues. FIG. 4C shows the 5′ and 31 border sequences of the SFV cDNA clone. The general outline how to obtain and demonstrate infectivity of the full-length SFV RNA is depicted in FIG. 6. The complete nucleotide sequence of the pSP6-SFV4 SP6 transcript together with the amino acid sequences of the nonstructural and the structural polyproteins is shown in FIG. 5.

[0107] Typically, about 5 μg of RNA per 100 ng of template was obtained using 10 units of polymerase, but the yield could be increased considerably by the use of more enzyme. The conditions slightly differ from those reported earlier for the production of infectious transcripts of alphaviruses (52) (47). A maximum production of RNA was obtained with rNTP concentrations at 1 mM. However, since infectivity also is dependent on the presence of a 5′cap structure optimal infectivity was obtained when the GTP concentration in the transcription reaction was halved. This drop had only a marginal effect on the amounts of RNA produced but raised the specific infectivity by a factor of 3 (data not shown).

[0108] The cDNA sequence shown in FIG. 5 has been used in the following examples. However, sequences having one or a few nucleotides, which differ from those shown in FIG. 5, could also be useful as vectors, even if these might be less efficient as illustrated above with the SFV cDNA sequence lacking the first 5′-G nucleotide in FIG. 5.

EXAMPLE 2

[0109] In this example the construction of SFV DNA expression vectors is disclosed.

[0110] The cDNA clone coding for the complete genome of SFV obtained in Example 1 was used to construct a SFV DNA expression vector by deletion of the coding region of the 26S structural genes to make way for heterologous inserts. However, the nonstructural coding region, which is required for the production of the nsP1-4 replicase complex is preserved. RNA replication is dependent on short 5′ (nt 1-247) (53, 54, 55) and 3′ (nt 11423-11441) sequence elements (56, 57), and therefore, also these had to be included in the vector construct, as had the 26S promoter just upstream of the C gene (17, 18).

[0111] As is shown in FIG. 7, first, the XbaI (6640)-NsiI (8927) fragment from the SFV cDNA clone pSP6-SFV4 from Example 1 was cloned into PGEM7Zf(+)(Promega Corp., Wl, USA) (Step A). From the resulting plasmid, pGEM7Zf(+)-SFV, the EcoRI fragment (SFV coordinates 7391 and 88746) was cloned into M13 mpl9 to insert a BamHI-XmaI-SmaI polylinker sequence immediately downstream from the 26S promoter site using site-directed mutagenesis (step B). Once the correct mutants had been verfied by sequencing from M13 ssDNA (single stranded), the EcoRI fragments were reinserted into pGEM7Zf(+)-SFV (step C) and then cloned back as XbaI-Nsλ fragments into pSP6-SFV4 (step D). To delete the major part of the cDNA region coding for the structural proteins of SFV, these plasmids were then cut with AsuII (7783) and NdeI (11033), blunted using Klenow fragment in the presence of all four nucleotides, and religated to create the final vectors designated pSFV1, pSFV2 and pSFV3, respectively (step E). The vectors retain the promoter region of the 26S subgenomic RNA and the last 49 amino acids of the E1 protein as well as the complete non-coding 3′ end of the SFV genome.

[0112] In the vectors the subgenomic (26S) protein coding portion has been replaced with a polylinker sequence allowing the insertional cloning of foreign cDNA sequences under the 26S promoter. As is shown in FIG. 8 these three vectors have the same basic cassette inserted downstream from the 26S promoter, i.e. a polylinker (BamHI-SmaI-XmaI) followed by a translational stop-codons in all three reading frames. The vectors differ as to the position where the polylinker cassette has been inserted. In pSFV1 the cassette is situated 31 bases downstream of the 26S transcription initiation site. The initiation motive of the capsid gene translation is identical to the consensus sequence (58). Therefore, this motive has been provided for in pSFV2, where it is placed immediately after the motive of the capsid gene. Finally, pSFV3 has the cassette placed immediately after the initiation codon (AUG) of the capsid gene. Sequencing primers (SP) needed for checking both ends of an insert have been designed to hybridize either to the 26S promoter region (SP1), or to the region following the stop codon cassette (SP2).

[0113] Note that the 26S promoter overlaps with the 3′-end of the nsP4 coding region. For pSFV2, the cloning site is positioned immediately after the translation initiation site of the SFV capsid gene. For pSFV3, the cloning site is positioned three nucleotides further downstream, i.e. immediately following to the initial AUG codon of the SFV capsid gene. The three translation stop codons following the polylinker are boxed. The downstream sequencing primer (SP1) overlaps with the 26S promoter, and the upstream sequencing primer (Sp2) overlaps the XmaIII site.

EXAMPLE 3

[0114] In this example an in vivo packaging system encompassing helper virus vector constructs is prepared.

[0115] The system allows SFV variants defective in structural protein functions, or recombinant RNAs derived from the expression vector construct obtained in Example 2, to be packaged into infectious virus particles. Thus, this system allows recombinant RNAs to be introduced into cells by normal infection. The helper vector, called pSFV-Helper1, is constructed by deleting the region between the restriction endonuclease sites AccI (308) and AccI (6399) of pSP6-SFV4 obtained in Example 1 by cutting and religation as shown in FIG. 7, step F. The vector retains the 5′ and 3′ signals needed for RNA replication. Since almost the complete nsP region of the Helper vector is deleted, RNA produced from this construct will not replicate in the cell due to the lack of a functional replicase complex. As is shown in FIG. 9, after transcription in vitro of pSFVl-recombinant and helper cDNAs, helper RNA is cotransfected with the pSFV1—recombinant derivative, the helper construct providing the structural proteins needed to assemble new virus particles, and the recombinant providing the nonstructural proteins needed for RNA replication, SFV particles comprising recombinant genomes being produced. The cotransfection is preferably produced by electroporation as is disclosed in Example 6 and preferably BHK cells are used as host cells.

[0116] To package the RNA a region at the end of nsP1 is required, an area which has been shown to bind capsid protein (57, 59). Since the Helper lacks this region, RNA derived from this vector will not be packaged and hence, transfections with recombinant and Helper produces only virus particles that carry recombinant-derived RNA. It follows that these viruses cannot be passaged further and thus provide a one-step virus stock. The advantage is that infections with these particles will not produce any viral proteins.

EXAMPLE 4

[0117] This example illustrates the construction of variants of the full-length SFV cDNA clone from Example 1 that allow insertion of foreign DNA sequences encoding foreign epitopes, and the production of recombinant (chimaeric) virus carrying said foreign epitopes as integral parts of the p62, E2 or E1 spike proteins. To this end, a thorough knowledge of the function, topology and antigenic structure of the E2 and E1 envelope proteins has been of the essence. Earlier studies on the pathogenicity of alphaviruses have shown that antibodies against E2 are type-specific and have good neutralizing activity while those against E1 generally are group-specific and are non-neutralizing (5). However, not until recently have antigenic sites of the closely related alphaviruses SFV, Sindbis, and Ross River been mapped and correlated to the level of amino acid sequence (60, 61, 62, 63). These studies have shown that the most dominant sites in question are at amino acid positions 216, 234 and 246-251 of the SFV E2 spike protein. Interestingly, these three sites are exactly the same as the ones predicted by computer analysis. In the present example domain 246-251 was used, since this area has a highly conserved structure and hydropathy profile within the group of alpha-viruses. Insertion of a gene encoding a foreign epitope into the 246-251 region of the pSP6-SFV4 p62 protein yields particles with one new epitope on each heterodimer, i.e. 240 copies.

[0118] To create a unique restriction endonuclease site that would allow specific insertion of foreign epitopes into the E2 portion of the SFV genome, a BamHI site was inserted by site directed mutagenesis using the oligonucleotide 5′-GATCGGCCTAGGAGCCGAGAGCCC.

EXAMPLE 5

[0119] In this example a conditionally lethal variant of SFV is constructed from the SFV cDNA obtained in Example 1, which variant carries a mutation in the p62 protein resulting in a noncleavable from of said protein, with the result that this variant as such cannot infect new host cells, unless first cleaved with exogenously added protease.

[0120] As illustrated in FIG. 10, this construct can be advantageously used as a vaccine carrier for foreign epitopes, since this form of the virus cannot enter new host cells although assembled with wild type efficiency in transfected cells. The block can be overcome by trypsin treatment of inactive virus particles. This converts the particle into a fully entry-competent form which can be used for amplification of this virus variant stock.

[0121] Once activated the SFV variant will enter cells normally through the endocytic pathway and start infection. Viral proteins will be made and budding takes place at the plasma membrane. However, all virus particles produced will be of inactive form and the infection will thus cease after one round of replication. The reason for the block in infection proficiency is a mutation which has been introduced by site directed mutagenesis into the cleavage site of p62. This arginine to leucine substitution (at amino acid postion 66 of the E3 portion of the p62 protein) changes the consensus features of the cleavage site so that it will not be recognized by the host cell proteinase that normally cleaves the p62 protein to the E2 and E3 polypeptides during transport to the cell surface. Instead, only exogenously added trypsin will be able to perform this cleavage, which in this case occurs at the arginine residue 65 immediately preceding the original cleavage site. As this cleavage regulates the activation of the entry function potential of the virus by controlling the binding of the entry spike subunit, the virus particle carrying only uncleaved p62 will be completely unable to enter new host cells. The creation of the cleavage deficient mutation E2 has been described earlier (29). An Asul1-Nsλ fragment spanning this region was then isolated and cloned into the full-length cDNA clonepSP6-SFV4.

EXAMPLE 6

[0122] In this example transfection of BHK cells with SFV RNA molecules transcribed in vitro from full-length cDNA from Example 1 or variants thereof or the SFV vectors from Example 2, which comprise exogenous DNA, is disclosed. The transfection is carried out by electroporation which is shown to be very efficient at optimized conditions.

[0123] BHK cells were transfected with the above SFV RNA molecules by electroporation and optimal conditions were determined by varying parameters like temperature, voltage, capacitance, and number of pulses. Optimal transfection was obtained by 2 consecutive pulses of 1.5 kV at 25 μF, under which negligible amounts of cells were killed. It was found that it was better to keep the cells at room tempeature than at 0° C. during the whole procedure. Transfection by electroporation was also measured as a function of input RNA. As expected, an increase in transfection frequency was not linearly dependent on RNA concentration, and about 2 μg of cRNA were needed to obtain 100% transfection.

[0124] On comparison with conventional transfection, this is a great improvement. For example, with DEAE-Dextran transfection optimally, only 0.2% of the cells were transfected.

EXAMPLE 7

[0125] This example illustrates heterologous gene expression driven by the SFV vector, pSFV1 from Example 2, for genes encoding the 21 kD cytoplasmic mouse dihydrofolate reductase (dhfr), the 90 kD membrane protein human transferrin receptor (TR), and finally the 14 kD secretory protein chicken lysozyme. The dhfr gene was isolated from pGEM2-dhfr (64) as a BamHI-HindIII fragment blunted with Klenow fragment and inserted into SmaI-cut pSFV1. The transferrin receptor gene was first cloned from pGEM1-TR (64, 65) as an XbaI-EcoRI fragment into pGEM7ZF(+) and subsequently from there as a BamHI fragment into pSFV1. Finally, a BamHI fragment from pGEM2 carrying the lysozyme gene (21) was cloned into pSFV1.

[0126] To study the expression of the heterologous proteins, in vitro-made RNA of the dhfr and TR constructs was electroporated into BHK cells. RNA of wild type SFV was used as control. At different time points post electroporation (p.e.) cells were pulse-labeled for 10 min followed by a 10 min chase, whereafter the lysates were analyzed by gel electrophoresis and autoradiography. The results are shown in FIG. 11. More specifically, BHK cells were transfected with RNAs of wild type SFV, pSFV1-dhfr, and pSFV1-TR, pulse-labeled at 3, 6, 9, 12, 15 and 24 h p.e. Equal amounts of lysate were run on a 12% gel. The 9 h sample was also used in immunoprecipitation (IP) of the SFV, the dhfr and the transferrin receptor proteins. Cells transfected with pSFV1-lysozyme were pulse-labeled at 9 h p.e. and then chased for the times (hours) indicated. An equal portion of lysate or medium was loaded on the 13.5% gel. IP represents immunoprecipitation from the 1 h chase lysate sample. The U-lane is lysate of labeled but untransfected cells. At 3 h p.e. hardly any exogenous proteins were made, since the incoming RNA starts with minus strand synthesis which does not peak until about 4-5 h p.e. (5). At this time point, almost all labeled proteins were of hos origin. In contrast, at 6 h p.e. the exogenous proteins were synthesized with great efficiency, and severe inhibition of host protein synthesis was evident. This was even more striking at 9 h p.e., when maximum shut down had been reached. Efficient production of the heteroloqous proteins continued up to 24 h p.e., after which production slowed down (data not shown), indicating that the cells had entered a stationary phase.

[0127] Since chicken lysozyme is a secretory protein, its expression was analyzed both from cell lysates and from the growth medium. Cells were pulse-labeled at 9 h p.e. and then chased up to 8 h. The results are shown in FIG. 11. Although lysozyme was slowly secreted, almost all labeled material was secreted to the medium during the chase.

EXAMPLE 8

[0128] This example illustrates the present in vivo packaging system.

[0129] In vitro-made RNA of pSFVl-TR was mixed with Helper RNA at different ratios and these mixtures were co-transfected into BHK cells. Cells were grown for 24 h after which the culture medium was collected and the virus particles pelleted by ultracentrifugation. The number of infectious units (i.u.) was determined by immunofluorescence. It was found that a 1:1 ratio of Helper and recombinant most efficiently produced infectious particles, and on the average 5×10⁶ cells yielded 2.5×10⁹ i.u. The infectivity of the virus stock was tested by infecting BHK cells at different multiplicities of infection (m.o.i.). In FIG. 11 the results for expression of human transferrin receptor in BHK cells after infection by such in vivo packaged particles carrying pSFV1-TR recombinant RNA is shown to the lower right. 200 μl of virus diluted in MEM (including 0.5% BAS and 2 mM glutamine) was overlaid on cells to give m.o.i. values ranging from 5 to 0.005. After 1 h at 37° C., complete BHK medium was added and growth continued for 9 h, at which time a 10 min pulse (100 μCi ³⁵S-methionine/ml) and 10 min chase was performed, and the cells dissolved in lysis buffer. 10 μl out of the 300 μl lysate (corresponding to 30,000 cells) was run on the 10% gel, and the dried gel was exposed for 2 h at −70° C. Due to the high expression level, only 3,000 cells are needed to obtain a distinct band on the autoradiograph with an over night exposure. Thus, it was found that efficient protein production and concomitant hos protein shut-off occurred at about 1 i.u. per cell. Since one SFV infected cell produces on the average 10⁸ capsid protein molecules, it follows that a virus stock produced from a single electroporation can be used to produce 10¹⁷ protein molecules equaling about 50 mg of protein.

[0130] From the foregoing experimental results it is obvious that the present invention is related to very useful and efficient expression system which lacks several of the disadvantages of the hitherto existing expression system. The major advantages of the present system are shortly summarized as follows:

[0131] (1) High titre recombinant virus stocks can be produced in one day by one transfection experiment. There is no need for selection/screening, plaque purification and amplification steps. This is appreciated since an easy production of recombinant virus is especially important in experiments where the phenotypes of large series of mutants have to be characterized.

[0132] (2) The recombinant virus stock is free from helper virus since only the recombinant genome but not the helper genome contains a packaging signal.

[0133] (3) The recombinant virus can be used to infect the recombinant genome in a “natural” and nonleakey way into a large variety of cells including insect and most higher euoaryotic cell types. Such a wide host range is very useful for an expressions system especially when cell-type-specific posttranslational modification reactions are required for the activity of the expressed protein.

[0134] (4) The level of protein expression obtained is extremely high, the level corresponding to those of the viral proteins during infection. There is also a host cell protein shut-off which makes it possible to follow the foreign proteins clearly in cell lysates without the need for antibody mediated antigen concentration. This will facilitate DNA expression experiments in cell biology considerably. Furthermore, problems of interference by the endogenous counter part to an expressed protein (i.e. homo-oligomerization reactions) can be avoided.

EXAMPLE 9

[0135] This example illustrates epitope carriers. A very important example where vaccine development is of the utmost importance concerns the acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus HIV-1 (66, 67). Sofar, all attempts to produce an efficient vaccine against HIV-1 have failed, although there was a very recent report that vaccination with disrupted SIV-1 (Simian immunodeficiency virus) to a certain extent may give protection against infections of that virus (68). However, development of safe and effective vaccine against HIV-1 will be very difficult due to the biological properties of the virus. In the present example, one epitope of HIV-1 was inserted into an antigenic domain of the E2 protein of SFV. The epitope used is located in glycoprotein gp120 of HIV-1, spanning amino acids 309-325. This forms the variable loop of HIV-1 and is situated immediately after an N-glycosylated site.

[0136] A chimaera was constructed where the 309-325 epitope of HIV was inserted into the BamHI site using cassette insertion of ready-made oligonucleotides encoding the HIV epitope. The required base substitutions at the BamHI site did not lead to any amino acid changes in the vector, although two amino acids (Asp and Glu) changed places. This change did not have any deleterious effect since in vitro made vector RNA induced cell infection with wild type efficiency. FIG. 12 shows the sequences in the area of interest in the epitope carrier. In preliminary experiments, it has been shown that chimaeric proteins were produced. The proteins can be immunoprecipitated with anti-HIV antibodies. It is to be expected that these are also used for production of chimaeric virus particles that can be used for vaccine preparation against HIV. Such particles are shown in FIG. 12, lower part.

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1 27 11517 base pairs nucleic acid single linear RNA (genomic) NO NO Semliki Forest Virus - 1..11517 /label= genome /note= “Semliki Forest Virus complete nucleotide sequence, presented as a cloned DNA sequence; see Figure 5.” CDS 87..7379 /product= “SFV polyprotein” CDS 7421..11179 /product= “SFV polyprotein” 1 GATGGCGGAT GTGTGACATA CACGACGCCA AAAGATTTTG TTCCAGCTCC TGCCACCTCC 60 GCTACGCGAG AGATTAACCA CCCACG ATG GCC GCC AAA GTG CAT GTT GAT ATT 113 Met Ala Ala Lys Val His Val Asp Ile 1 5 GAG GCT GAC AGC CCA TTC ATC AAG TCT TTG CAG AAG GCA TTT CCG TCG 161 Glu Ala Asp Ser Pro Phe Ile Lys Ser Leu Gln Lys Ala Phe Pro Ser 10 15 20 25 TTC GAG GTG GAG TCA TTG CAG GTC ACA CCA AAT GAC CAT GCA AAT GCC 209 Phe Glu Val Glu Ser Leu Gln Val Thr Pro Asn Asp His Ala Asn Ala 30 35 40 AGA GCA TTT TCG CAC CTG GCT ACC AAA TTG ATC GAG CAG GAG ACT GAC 257 Arg Ala Phe Ser His Leu Ala Thr Lys Leu Ile Glu Gln Glu Thr Asp 45 50 55 AAA GAC ACA CTC ATC TTG GAT ATC GGC AGT GCG CCT TCC AGG AGA ATG 305 Lys Asp Thr Leu Ile Leu Asp Ile Gly Ser Ala Pro Ser Arg Arg Met 60 65 70 ATG TCT ACG CAC AAA TAC CAC TGC GTA TGC CCT ATG CGC AGC GCA GAA 353 Met Ser Thr His Lys Tyr His Cys Val Cys Pro Met Arg Ser Ala Glu 75 80 85 GAC CCC GAA AGG CTC GAT AGC TAC GCA AAG AAA CTG GCA GCG GCC TCC 401 Asp Pro Glu Arg Leu Asp Ser Tyr Ala Lys Lys Leu Ala Ala Ala Ser 90 95 100 105 GGG AAG GTG CTG GAT AGA GAG ATC GCA GGA AAA ATC ACC GAC CTG CAG 449 Gly Lys Val Leu Asp Arg Glu Ile Ala Gly Lys Ile Thr Asp Leu Gln 110 115 120 ACC GTC ATG GCT ACG CCA GAC GCT GAA TCT CCT ACC TTT TGC CTG CAT 497 Thr Val Met Ala Thr Pro Asp Ala Glu Ser Pro Thr Phe Cys Leu His 125 130 135 ACA GAC GTC ACG TGT CGT ACG GCA GCC GAA GTG GCC GTA TAC CAG GAC 545 Thr Asp Val Thr Cys Arg Thr Ala Ala Glu Val Ala Val Tyr Gln Asp 140 145 150 GTG TAT GCT GTA CAT GCA CCA ACA TCG CTG TAC CAT CAG GCG ATG AAA 593 Val Tyr Ala Val His Ala Pro Thr Ser Leu Tyr His Gln Ala Met Lys 155 160 165 GGT GTC AGA ACG GCG TAT TGG ATT GGG TTT GAC ACC ACC CCG TTT ATG 641 Gly Val Arg Thr Ala Tyr Trp Ile Gly Phe Asp Thr Thr Pro Phe Met 170 175 180 185 TTT GAC GCG CTA GCA GGC GCG TAT CCA ACC TAC GCC ACA AAC TGG GCC 689 Phe Asp Ala Leu Ala Gly Ala Tyr Pro Thr Tyr Ala Thr Asn Trp Ala 190 195 200 GAC GAG CAG GTG TTA CAG GCC AGG AAC ATA GGA CTG TGT GCA GCA TCC 737 Asp Glu Gln Val Leu Gln Ala Arg Asn Ile Gly Leu Cys Ala Ala Ser 205 210 215 TTG ACT GAG GGA AGA CTC GGC AAA CTG TCC ATT CTC CGC AAG AAG CAA 785 Leu Thr Glu Gly Arg Leu Gly Lys Leu Ser Ile Leu Arg Lys Lys Gln 220 225 230 TTG AAA CCT TGC GAC ACA GTC ATG TTC TCG GTA GGA TCT ACA TTG TAC 833 Leu Lys Pro Cys Asp Thr Val Met Phe Ser Val Gly Ser Thr Leu Tyr 235 240 245 ACT GAG AGC AGA AAG CTA CTG AGG AGC TGG CAC TTA CCC TCC GTA TTC 881 Thr Glu Ser Arg Lys Leu Leu Arg Ser Trp His Leu Pro Ser Val Phe 250 255 260 265 CAC CTG AAA GGT AAA CAA TCC TTT ACC TGT AGG TGC GAT ACC ATC GTA 929 His Leu Lys Gly Lys Gln Ser Phe Thr Cys Arg Cys Asp Thr Ile Val 270 275 280 TCA TGT GAA GGG TAC GTA GTT AAG AAA ATC ACT ATG TGC CCC GGC CTG 977 Ser Cys Glu Gly Tyr Val Val Lys Lys Ile Thr Met Cys Pro Gly Leu 285 290 295 TAC GGT AAA ACG GTA GGG TAC GCC GTG ACG TAT CAC GCG GAG GGA TTC 1025 Tyr Gly Lys Thr Val Gly Tyr Ala Val Thr Tyr His Ala Glu Gly Phe 300 305 310 CTA GTG TGC AAG ACC ACA GAC ACT GTC AAA GGA GAA AGA GTC TCA TTC 1073 Leu Val Cys Lys Thr Thr Asp Thr Val Lys Gly Glu Arg Val Ser Phe 315 320 325 CCT GTA TGC ACC TAC GTC CCC TCA ACC ATC TGT GAT CAA ATG ACT GGC 1121 Pro Val Cys Thr Tyr Val Pro Ser Thr Ile Cys Asp Gln Met Thr Gly 330 335 340 345 ATA CTA GCG ACC GAC GTC ACA CCG GAG GAC GCA CAG AAG TTG TTA GTG 1169 Ile Leu Ala Thr Asp Val Thr Pro Glu Asp Ala Gln Lys Leu Leu Val 350 355 360 GGA TTG AAT CAG AGG ATA GTT GTG AAC GGA AGA ACA CAG CGA AAC ACT 1217 Gly Leu Asn Gln Arg Ile Val Val Asn Gly Arg Thr Gln Arg Asn Thr 365 370 375 AAC ACG ATG AAG AAC TAT CTG CTT CCG ATT GTG GCC GTC GCA TTT AGC 1265 Asn Thr Met Lys Asn Tyr Leu Leu Pro Ile Val Ala Val Ala Phe Ser 380 385 390 AAG TGG GCG AGG GAA TAC AAG GCA GAC CTT GAT GAT GAA AAA CCT CTG 1313 Lys Trp Ala Arg Glu Tyr Lys Ala Asp Leu Asp Asp Glu Lys Pro Leu 395 400 405 GGT GTC CGA GAG AGG TCA CTT ACT TGC TGC TGC TTG TGG GCA TTT AAA 1361 Gly Val Arg Glu Arg Ser Leu Thr Cys Cys Cys Leu Trp Ala Phe Lys 410 415 420 425 ACG AGG AAG ATG CAC ACC ATG TAC AAG AAA CCA GAC ACC CAG ACA ATA 1409 Thr Arg Lys Met His Thr Met Tyr Lys Lys Pro Asp Thr Gln Thr Ile 430 435 440 GTG AAG GTG CCT TCA GAG TTT AAC TCG TTC GTC ATC CCG AGC CTA TGG 1457 Val Lys Val Pro Ser Glu Phe Asn Ser Phe Val Ile Pro Ser Leu Trp 445 450 455 TCT ACA GGC CTC GCA ATC CCA GTC AGA TCA CGC ATT AAG ATG CTT TTG 1505 Ser Thr Gly Leu Ala Ile Pro Val Arg Ser Arg Ile Lys Met Leu Leu 460 465 470 GCC AAG AAG ACC AAG CGA GAG TTA ATA CCT GTT CTC GAC GCG TCG TCA 1553 Ala Lys Lys Thr Lys Arg Glu Leu Ile Pro Val Leu Asp Ala Ser Ser 475 480 485 GCC AGG GAT GCT GAA CAA GAG GAG AAG GAG AGG TTG GAG GCC GAG CTG 1601 Ala Arg Asp Ala Glu Gln Glu Glu Lys Glu Arg Leu Glu Ala Glu Leu 490 495 500 505 ACT AGA GAA GCC TTA CCA CCC CTC GTC CCC ATC GCG CCG GCG GAG ACG 1649 Thr Arg Glu Ala Leu Pro Pro Leu Val Pro Ile Ala Pro Ala Glu Thr 510 515 520 GGA GTC GTC GAC GTC GAC GTT GAA GAA CTA GAG TAT CAC GCA GGT GCA 1697 Gly Val Val Asp Val Asp Val Glu Glu Leu Glu Tyr His Ala Gly Ala 525 530 535 GGG GTC GTG GAA ACA CCT CGC AGC GCG TTG AAA GTC ACC GCA CAG CCG 1745 Gly Val Val Glu Thr Pro Arg Ser Ala Leu Lys Val Thr Ala Gln Pro 540 545 550 AAC GAC GTA CTA CTA GGA AAT TAC GTA GTT CTG TCC CCG CAG ACC GTG 1793 Asn Asp Val Leu Leu Gly Asn Tyr Val Val Leu Ser Pro Gln Thr Val 555 560 565 CTC AAG AGC TCC AAG TTG GCC CCC GTG CAC CCT CTA GCA GAG CAG GTG 1841 Leu Lys Ser Ser Lys Leu Ala Pro Val His Pro Leu Ala Glu Gln Val 570 575 580 585 AAA ATA ATA ACA CAT AAC GGG AGG GCC GGC GGT TAC CAG GTC GAC GGA 1889 Lys Ile Ile Thr His Asn Gly Arg Ala Gly Gly Tyr Gln Val Asp Gly 590 595 600 TAT GAC GGC AGG GTC CTA CTA CCA TGT GGA TCG GCC ATT CCG GTC CCT 1937 Tyr Asp Gly Arg Val Leu Leu Pro Cys Gly Ser Ala Ile Pro Val Pro 605 610 615 GAG TTT CAA GCT TTG AGC GAG AGC GCC ACT ATG GTG TAC AAC GAA AGG 1985 Glu Phe Gln Ala Leu Ser Glu Ser Ala Thr Met Val Tyr Asn Glu Arg 620 625 630 GAG TTC GTC AAC AGG AAA CTA TAC CAT ATT GCC GTT CAC GGA CCG TCG 2033 Glu Phe Val Asn Arg Lys Leu Tyr His Ile Ala Val His Gly Pro Ser 635 640 645 CTG AAC ACC GAC GAG GAG AAC TAC GAG AAA GTC AGA GCT GAA AGA ACT 2081 Leu Asn Thr Asp Glu Glu Asn Tyr Glu Lys Val Arg Ala Glu Arg Thr 650 655 660 665 GAC GCC GAG TAC GTG TTC GAC GTA GAT AAA AAA TGC TGC GTC AAG AGA 2129 Asp Ala Glu Tyr Val Phe Asp Val Asp Lys Lys Cys Cys Val Lys Arg 670 675 680 GAG GAA GCG TCG GGT TTG GTG TTG GTG GGA GAG CTA ACC AAC CCC CCG 2177 Glu Glu Ala Ser Gly Leu Val Leu Val Gly Glu Leu Thr Asn Pro Pro 685 690 695 TTC CAT GAA TTC GCC TAC GAA GGG CTG AAG ATC AGG CCG TCG GCA CCA 2225 Phe His Glu Phe Ala Tyr Glu Gly Leu Lys Ile Arg Pro Ser Ala Pro 700 705 710 TAT AAG ACT ACA GTA GTA GGA GTC TTT GGG GTT CCG GGA TCA GGC AAG 2273 Tyr Lys Thr Thr Val Val Gly Val Phe Gly Val Pro Gly Ser Gly Lys 715 720 725 TCT GCT ATT ATT AAG AGC CTC GTG ACC AAA CAC GAT CTG GTC ACC AGC 2321 Ser Ala Ile Ile Lys Ser Leu Val Thr Lys His Asp Leu Val Thr Ser 730 735 740 745 GGC AAG AAG GAG AAC TGC CAG GAA ATA GTT AAC GAC GTG AAG AAG CAC 2369 Gly Lys Lys Glu Asn Cys Gln Glu Ile Val Asn Asp Val Lys Lys His 750 755 760 CGC GGG AAG GGG ACA AGT AGG GAA AAC AGT GAC TCC ATC CTG CTA AAC 2417 Arg Gly Lys Gly Thr Ser Arg Glu Asn Ser Asp Ser Ile Leu Leu Asn 765 770 775 GGG TGT CGT CGT GCC GTG GAC ATC CTA TAT GTG GAC GAG GCT TTC GCT 2465 Gly Cys Arg Arg Ala Val Asp Ile Leu Tyr Val Asp Glu Ala Phe Ala 780 785 790 TGC CAT TCC GGT ACT CTG CTG GCC CTA ATT GCT CTT GTT AAA CCT CGG 2513 Cys His Ser Gly Thr Leu Leu Ala Leu Ile Ala Leu Val Lys Pro Arg 795 800 805 AGC AAA GTG GTG TTA TGC GGA GAC CCC AAG CAA TGC GGA TTC TTC AAT 2561 Ser Lys Val Val Leu Cys Gly Asp Pro Lys Gln Cys Gly Phe Phe Asn 810 815 820 825 ATG ATG CAG CTT AAG GTG AAC TTC AAC CAC AAC ATC TGC ACT GAA GTA 2609 Met Met Gln Leu Lys Val Asn Phe Asn His Asn Ile Cys Thr Glu Val 830 835 840 TGT CAT AAA AGT ATA TCC AGA CGT TGC ACG CGT CCA GTC ACG GCC ATC 2657 Cys His Lys Ser Ile Ser Arg Arg Cys Thr Arg Pro Val Thr Ala Ile 845 850 855 GTG TCT ACG TTG CAC TAC GGA GGC AAG ATG CGC ACG ACC AAC CCG TGC 2705 Val Ser Thr Leu His Tyr Gly Gly Lys Met Arg Thr Thr Asn Pro Cys 860 865 870 AAC AAA CCC ATA ATC ATA GAC ACC ACA GGA CAG ACC AAG CCC AAG CCA 2753 Asn Lys Pro Ile Ile Ile Asp Thr Thr Gly Gln Thr Lys Pro Lys Pro 875 880 885 GGA GAC ATC GTG TTA ACA TGC TTC CGA GGC TGG GCA AAG CAG CTG CAG 2801 Gly Asp Ile Val Leu Thr Cys Phe Arg Gly Trp Ala Lys Gln Leu Gln 890 895 900 905 TTG GAC TAC CGT GGA CAC GAA GTC ATG ACA GCA GCA GCA TCT CAG GGC 2849 Leu Asp Tyr Arg Gly His Glu Val Met Thr Ala Ala Ala Ser Gln Gly 910 915 920 CTC ACC CGC AAA GGG GTA TAC GCC GTA AGG CAG AAG GTG AAT GAA AAT 2897 Leu Thr Arg Lys Gly Val Tyr Ala Val Arg Gln Lys Val Asn Glu Asn 925 930 935 CCC TTG TAT GCC CCT GCG TCG GAG CAC GTG AAT GTA CTG CTG ACG CGC 2945 Pro Leu Tyr Ala Pro Ala Ser Glu His Val Asn Val Leu Leu Thr Arg 940 945 950 ACT GAG GAT AGG CTG GTG TGG AAA ACG CTG GCC GGC GAT CCC TGG ATT 2993 Thr Glu Asp Arg Leu Val Trp Lys Thr Leu Ala Gly Asp Pro Trp Ile 955 960 965 AAG GTC CTA TCA AAC ATT CCA CAG GGT AAC TTT ACG GCC ACA TTG GAA 3041 Lys Val Leu Ser Asn Ile Pro Gln Gly Asn Phe Thr Ala Thr Leu Glu 970 975 980 985 GAA TGG CAA GAA GAA CAC GAC AAA ATA ATG AAG GTG ATT GAA GGA CCG 3089 Glu Trp Gln Glu Glu His Asp Lys Ile Met Lys Val Ile Glu Gly Pro 990 995 1000 GCT GCG CCT GTG GAC GCG TTC CAG AAC AAA GCG AAC GTG TGT TGG GCG 3137 Ala Ala Pro Val Asp Ala Phe Gln Asn Lys Ala Asn Val Cys Trp Ala 1005 1010 1015 AAA AGC CTG GTG CCT GTC CTG GAC ACT GCC GGA ATC AGA TTG ACA GCA 3185 Lys Ser Leu Val Pro Val Leu Asp Thr Ala Gly Ile Arg Leu Thr Ala 1020 1025 1030 GAG GAG TGG AGC ACC ATA ATT ACA GCA TTT AAG GAG GAC AGA GCT TAC 3233 Glu Glu Trp Ser Thr Ile Ile Thr Ala Phe Lys Glu Asp Arg Ala Tyr 1035 1040 1045 TCT CCA GTG GTG GCC TTG AAT GAA ATT TGC ACC AAG TAC TAT GGA GTT 3281 Ser Pro Val Val Ala Leu Asn Glu Ile Cys Thr Lys Tyr Tyr Gly Val 1050 1055 1060 1065 GAC CTG GAC AGT GGC CTG TTT TCT GCC CCG AAG GTG TCC CTG TAT TAC 3329 Asp Leu Asp Ser Gly Leu Phe Ser Ala Pro Lys Val Ser Leu Tyr Tyr 1070 1075 1080 GAG AAC AAC CAC TGG GAT AAC AGA CCT GGT GGA AGG ATG TAT GGA TTC 3377 Glu Asn Asn His Trp Asp Asn Arg Pro Gly Gly Arg Met Tyr Gly Phe 1085 1090 1095 AAT GCC GCA ACA GCT GCC AGG CTG GAA GCT AGA CAT ACC TTC CTG AAG 3425 Asn Ala Ala Thr Ala Ala Arg Leu Glu Ala Arg His Thr Phe Leu Lys 1100 1105 1110 GGG CAG TGG CAT ACG GGC AAG CAG GCA GTT ATC GCA GAA AGA AAA ATC 3473 Gly Gln Trp His Thr Gly Lys Gln Ala Val Ile Ala Glu Arg Lys Ile 1115 1120 1125 CAA CCG CTT TCT GTG CTG GAC AAT GTA ATT CCT ATC AAC CGC AGG CTG 3521 Gln Pro Leu Ser Val Leu Asp Asn Val Ile Pro Ile Asn Arg Arg Leu 1130 1135 1140 1145 CCG CAC GCC CTG GTG GCT GAG TAC AAG ACG GTT AAA GGC AGT AGG GTT 3569 Pro His Ala Leu Val Ala Glu Tyr Lys Thr Val Lys Gly Ser Arg Val 1150 1155 1160 GAG TGG CTG GTC AAT AAA GTA AGA GGG TAC CAC GTC CTG CTG GTG AGT 3617 Glu Trp Leu Val Asn Lys Val Arg Gly Tyr His Val Leu Leu Val Ser 1165 1170 1175 GAG TAC AAC CTG GCT TTG CCT CGA CGC AGG GTC ACT TGG TTG TCA CCG 3665 Glu Tyr Asn Leu Ala Leu Pro Arg Arg Arg Val Thr Trp Leu Ser Pro 1180 1185 1190 CTG AAT GTC ACA GGC GCC GAT AGG TGC TAC GAC CTA AGT TTA GGA CTG 3713 Leu Asn Val Thr Gly Ala Asp Arg Cys Tyr Asp Leu Ser Leu Gly Leu 1195 1200 1205 CCG GCT GAC GCC GGC AGG TTC GAC TTG GTC TTT GTG AAC ATT CAC ACG 3761 Pro Ala Asp Ala Gly Arg Phe Asp Leu Val Phe Val Asn Ile His Thr 1210 1215 1220 1225 GAA TTC AGA ATC CAC CAC TAC CAG CAG TGT GTC GAC CAC GCC ATG AAG 3809 Glu Phe Arg Ile His His Tyr Gln Gln Cys Val Asp His Ala Met Lys 1230 1235 1240 CTG CAG ATG CTT GGG GGA GAT GCG CTA CGA CTG CTA AAA CCC GGC GGC 3857 Leu Gln Met Leu Gly Gly Asp Ala Leu Arg Leu Leu Lys Pro Gly Gly 1245 1250 1255 ATC TTG ATG AGA GCT TAC GGA TAC GCC GAT AAA ATC AGC GAA GCC GTT 3905 Ile Leu Met Arg Ala Tyr Gly Tyr Ala Asp Lys Ile Ser Glu Ala Val 1260 1265 1270 GTT TCC TCC TTA AGC AGA AAG TTC TCG TCT GCA AGA GTG TTG CGC CCG 3953 Val Ser Ser Leu Ser Arg Lys Phe Ser Ser Ala Arg Val Leu Arg Pro 1275 1280 1285 GAT TGT GTC ACC AGC AAT ACA GAA GTG TTC TTG CTG TTC TCC AAC TTT 4001 Asp Cys Val Thr Ser Asn Thr Glu Val Phe Leu Leu Phe Ser Asn Phe 1290 1295 1300 1305 GAC AAC GGA AAG AGA CCC TCT ACG CTA CAC CAG ATG AAT ACC AAG CTG 4049 Asp Asn Gly Lys Arg Pro Ser Thr Leu His Gln Met Asn Thr Lys Leu 1310 1315 1320 AGT GCC GTG TAT GCC GGA GAA GCC ATG CAC ACG GCC GGG TGT GCA CCA 4097 Ser Ala Val Tyr Ala Gly Glu Ala Met His Thr Ala Gly Cys Ala Pro 1325 1330 1335 TCC TAC AGA GTT AAG AGA GCA GAC ATA GCC ACG TGC ACA GAA GCG GCT 4145 Ser Tyr Arg Val Lys Arg Ala Asp Ile Ala Thr Cys Thr Glu Ala Ala 1340 1345 1350 GTG GTT AAC GCA GCT AAC GCC CGT GGA ACT GTA GGG GAT GGC GTA TGC 4193 Val Val Asn Ala Ala Asn Ala Arg Gly Thr Val Gly Asp Gly Val Cys 1355 1360 1365 AGG GCC GTG GCG AAG AAA TGG CCG TCA GCC TTT AAG GGA GCA GCA ACA 4241 Arg Ala Val Ala Lys Lys Trp Pro Ser Ala Phe Lys Gly Ala Ala Thr 1370 1375 1380 1385 CCA GTG GGC ACA ATT AAA ACA GTC ATG TGC GGC TCG TAC CCC GTC ATC 4289 Pro Val Gly Thr Ile Lys Thr Val Met Cys Gly Ser Tyr Pro Val Ile 1390 1395 1400 CAC GCT GTA GCG CCT AAT TTC TCT GCC ACG ACT GAA GCG GAA GGG GAC 4337 His Ala Val Ala Pro Asn Phe Ser Ala Thr Thr Glu Ala Glu Gly Asp 1405 1410 1415 CGC GAA TTG GCC GCT GTC TAC CGG GCA GTG GCC GCC GAA GTA AAC AGA 4385 Arg Glu Leu Ala Ala Val Tyr Arg Ala Val Ala Ala Glu Val Asn Arg 1420 1425 1430 CTG TCA CTG AGC AGC GTA GCC ATC CCG CTG CTG TCC ACA GGA GTG TTC 4433 Leu Ser Leu Ser Ser Val Ala Ile Pro Leu Leu Ser Thr Gly Val Phe 1435 1440 1445 AGC GGC GGA AGA GAT AGG CTG CAG CAA TCC CTC AAC CAT CTA TTC ACA 4481 Ser Gly Gly Arg Asp Arg Leu Gln Gln Ser Leu Asn His Leu Phe Thr 1450 1455 1460 1465 GCA ATG GAC GCC ACG GAC GCT GAC GTG ACC ATC TAC TGC AGA GAC AAA 4529 Ala Met Asp Ala Thr Asp Ala Asp Val Thr Ile Tyr Cys Arg Asp Lys 1470 1475 1480 AGT TGG GAG AAG AAA ATC CAG GAA GCC ATT GAC ATG AGG ACG GCT GTG 4577 Ser Trp Glu Lys Lys Ile Gln Glu Ala Ile Asp Met Arg Thr Ala Val 1485 1490 1495 GAG TTG CTC AAT GAT GAC GTG GAG CTG ACC ACA GAC TTG GTG AGA GTG 4625 Glu Leu Leu Asn Asp Asp Val Glu Leu Thr Thr Asp Leu Val Arg Val 1500 1505 1510 CAC CCG GAC AGC AGC CTG GTG GGT CGT AAG GGC TAC AGT ACC ACT GAC 4673 His Pro Asp Ser Ser Leu Val Gly Arg Lys Gly Tyr Ser Thr Thr Asp 1515 1520 1525 GGG TCG CTG TAC TCG TAC TTT GAA GGT ACG AAA TTC AAC CAG GCT GCT 4721 Gly Ser Leu Tyr Ser Tyr Phe Glu Gly Thr Lys Phe Asn Gln Ala Ala 1530 1535 1540 1545 ATT GAT ATG GCA GAG ATA CTG ACG TTG TGG CCC AGA CTG CAA GAG GCA 4769 Ile Asp Met Ala Glu Ile Leu Thr Leu Trp Pro Arg Leu Gln Glu Ala 1550 1555 1560 AAC GAA CAG ATA TGC CTA TAC GCG CTG GGC GAA ACA ATG GAC AAC ATC 4817 Asn Glu Gln Ile Cys Leu Tyr Ala Leu Gly Glu Thr Met Asp Asn Ile 1565 1570 1575 AGA TCC AAA TGT CCG GTG AAC GAT TCC GAT TCA TCA ACA CCT CCC AGG 4865 Arg Ser Lys Cys Pro Val Asn Asp Ser Asp Ser Ser Thr Pro Pro Arg 1580 1585 1590 ACA GTG CCC TGC CTG TGC CGC TAC GCA ATG ACA GCA GAA CGG ATC GCC 4913 Thr Val Pro Cys Leu Cys Arg Tyr Ala Met Thr Ala Glu Arg Ile Ala 1595 1600 1605 CGC CTT AGG TCA CAC CAA GTT AAA AGC ATG GTG GTT TGC TCA TCT TTT 4961 Arg Leu Arg Ser His Gln Val Lys Ser Met Val Val Cys Ser Ser Phe 1610 1615 1620 1625 CCC CTC CCG AAA TAC CAT GTA GAT GGG GTG CAG AAG GTA AAG TGC GAG 5009 Pro Leu Pro Lys Tyr His Val Asp Gly Val Gln Lys Val Lys Cys Glu 1630 1635 1640 AAG GTT CTC CTG TTC GAC CCG ACG GTA CCT TCA GTG GTT AGT CCG CGG 5057 Lys Val Leu Leu Phe Asp Pro Thr Val Pro Ser Val Val Ser Pro Arg 1645 1650 1655 AAG TAT GCC GCA TCT ACG ACG GAC CAC TCA GAT CGG TCG TTA CGA GGG 5105 Lys Tyr Ala Ala Ser Thr Thr Asp His Ser Asp Arg Ser Leu Arg Gly 1660 1665 1670 TTT GAC TTG GAC TGG ACC ACC GAC TCG TCT TCC ACT GCC AGC GAT ACC 5153 Phe Asp Leu Asp Trp Thr Thr Asp Ser Ser Ser Thr Ala Ser Asp Thr 1675 1680 1685 ATG TCG CTA CCC AGT TTG CAG TCG TGT GAC ATC GAC TCG ATC TAC GAG 5201 Met Ser Leu Pro Ser Leu Gln Ser Cys Asp Ile Asp Ser Ile Tyr Glu 1690 1695 1700 1705 CCA ATG GCT CCC ATA GTA GTG ACG GCT GAC GTA CAC CCT GAA CCC GCA 5249 Pro Met Ala Pro Ile Val Val Thr Ala Asp Val His Pro Glu Pro Ala 1710 1715 1720 GGC ATC GCG GAC CTG GCG GCA GAT GTG CAC CCT GAA CCC GCA GAC CAT 5297 Gly Ile Ala Asp Leu Ala Ala Asp Val His Pro Glu Pro Ala Asp His 1725 1730 1735 GTG GAC CTC GAG AAC CCG ATT CCT CCA CCG CGC CCG AAG AGA GCT GCA 5345 Val Asp Leu Glu Asn Pro Ile Pro Pro Pro Arg Pro Lys Arg Ala Ala 1740 1745 1750 TAC CTT GCC TCC CGC GCG GCG GAG CGA CCG GTG CCG GCG CCG AGA AAG 5393 Tyr Leu Ala Ser Arg Ala Ala Glu Arg Pro Val Pro Ala Pro Arg Lys 1755 1760 1765 CCG ACG CCT GCC CCA AGG ACT GCG TTT AGG AAC AAG CTG CCT TTG ACG 5441 Pro Thr Pro Ala Pro Arg Thr Ala Phe Arg Asn Lys Leu Pro Leu Thr 1770 1775 1780 1785 TTC GGC GAC TTT GAC GAG CAC GAG GTC GAT GCG TTG GCC TCC GGG ATT 5489 Phe Gly Asp Phe Asp Glu His Glu Val Asp Ala Leu Ala Ser Gly Ile 1790 1795 1800 ACT TTC GGA GAC TTC GAC GAC GTC CTG CGA CTA GGC CGC GCG GGT GCA 5537 Thr Phe Gly Asp Phe Asp Asp Val Leu Arg Leu Gly Arg Ala Gly Ala 1805 1810 1815 TAT ATT TTC TCC TCG GAC ACT GGC AGC GGA CAT TTA CAA CAA AAA TCC 5585 Tyr Ile Phe Ser Ser Asp Thr Gly Ser Gly His Leu Gln Gln Lys Ser 1820 1825 1830 GTT AGG CAG CAC AAT CTC CAG TGC GCA CAA CTG GAT GCG GTC CAG GAG 5633 Val Arg Gln His Asn Leu Gln Cys Ala Gln Leu Asp Ala Val Gln Glu 1835 1840 1845 GAG AAA ATG TAC CCG CCA AAA TTG GAT ACT GAG AGG GAG AAG CTG TTG 5681 Glu Lys Met Tyr Pro Pro Lys Leu Asp Thr Glu Arg Glu Lys Leu Leu 1850 1855 1860 1865 CTG CTG AAA ATG CAG ATG CAC CCA TCG GAG GCT AAT AAG AGT CGA TAC 5729 Leu Leu Lys Met Gln Met His Pro Ser Glu Ala Asn Lys Ser Arg Tyr 1870 1875 1880 CAG TCT CGC AAA GTG GAG AAC ATG AAA GCC ACG GTG GTG GAC AGG CTC 5777 Gln Ser Arg Lys Val Glu Asn Met Lys Ala Thr Val Val Asp Arg Leu 1885 1890 1895 ACA TCG GGG GCC AGA TTG TAC ACG GGA GCG GAC GTA GGC CGC ATA CCA 5825 Thr Ser Gly Ala Arg Leu Tyr Thr Gly Ala Asp Val Gly Arg Ile Pro 1900 1905 1910 ACA TAC GCG GTT CGG TAC CCC CGC CCC GTG TAC TCC CCT ACC GTG ATC 5873 Thr Tyr Ala Val Arg Tyr Pro Arg Pro Val Tyr Ser Pro Thr Val Ile 1915 1920 1925 GAA AGA TTC TCA AGC CCC GAT GTA GCA ATC GCA GCG TGC AAC GAA TAC 5921 Glu Arg Phe Ser Ser Pro Asp Val Ala Ile Ala Ala Cys Asn Glu Tyr 1930 1935 1940 1945 CTA TCC AGA AAT TAC CCA ACA GTG GCG TCG TAC CAG ATA ACA GAT GAA 5969 Leu Ser Arg Asn Tyr Pro Thr Val Ala Ser Tyr Gln Ile Thr Asp Glu 1950 1955 1960 TAC GAC GCA TAC TTG GAC ATG GTT GAC GGG TCG GAT AGT TGC TTG GAC 6017 Tyr Asp Ala Tyr Leu Asp Met Val Asp Gly Ser Asp Ser Cys Leu Asp 1965 1970 1975 AGA GCG ACA TTC TGC CCG GCG AAG CTC CGG TGC TAC CCG AAA CAT CAT 6065 Arg Ala Thr Phe Cys Pro Ala Lys Leu Arg Cys Tyr Pro Lys His His 1980 1985 1990 GCG TAC CAC CAG CCG ACT GTA CGC AGT GCC GTC CCG TCA CCC TTT CAG 6113 Ala Tyr His Gln Pro Thr Val Arg Ser Ala Val Pro Ser Pro Phe Gln 1995 2000 2005 AAC ACA CTA CAG AAC GTG CTA GCG GCC GCC ACC AAG AGA AAC TGC AAC 6161 Asn Thr Leu Gln Asn Val Leu Ala Ala Ala Thr Lys Arg Asn Cys Asn 2010 2015 2020 2025 GTC ACG CAA ATG CGA GAA CTA CCC ACC ATG GAC TCG GCA GTG TTC AAC 6209 Val Thr Gln Met Arg Glu Leu Pro Thr Met Asp Ser Ala Val Phe Asn 2030 2035 2040 GTG GAG TGC TTC AAG CGC TAT GCC TGC TCC GGA GAA TAT TGG GAA GAA 6257 Val Glu Cys Phe Lys Arg Tyr Ala Cys Ser Gly Glu Tyr Trp Glu Glu 2045 2050 2055 TAT GCT AAA CAA CCT ATC CGG ATA ACC ACT GAG AAC ATC ACT ACC TAT 6305 Tyr Ala Lys Gln Pro Ile Arg Ile Thr Thr Glu Asn Ile Thr Thr Tyr 2060 2065 2070 GTG ACC AAA TTG AAA GGC CCG AAA GCT GCT GCC TTG TTC GCT AAG ACC 6353 Val Thr Lys Leu Lys Gly Pro Lys Ala Ala Ala Leu Phe Ala Lys Thr 2075 2080 2085 CAC AAC TTG GTT CCG CTG CAG GAG GTT CCC ATG GAC AGA TTC ACG GTC 6401 His Asn Leu Val Pro Leu Gln Glu Val Pro Met Asp Arg Phe Thr Val 2090 2095 2100 2105 GAC ATG AAA CGA GAT GTC AAA GTC ACT CCA GGG ACG AAA CAC ACA GAG 6449 Asp Met Lys Arg Asp Val Lys Val Thr Pro Gly Thr Lys His Thr Glu 2110 2115 2120 GAA AGA CCC AAA GTC CAG GTA ATT CAA GCA GCG GAG CCA TTG GCG ACC 6497 Glu Arg Pro Lys Val Gln Val Ile Gln Ala Ala Glu Pro Leu Ala Thr 2125 2130 2135 GCT TAC CTG TGC GGC ATC CAC AGG GAA TTA GTA AGG AGA CTA AAT GCT 6545 Ala Tyr Leu Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala 2140 2145 2150 GTG TTA CGC CCT AAC GTG CAC ACA TTG TTT GAT ATG TCG GCC GAA GAC 6593 Val Leu Arg Pro Asn Val His Thr Leu Phe Asp Met Ser Ala Glu Asp 2155 2160 2165 TTT GAC GCG ATC ATC GCC TCT CAC TTC CAC CCA GGA GAC CCG GTT CTA 6641 Phe Asp Ala Ile Ile Ala Ser His Phe His Pro Gly Asp Pro Val Leu 2170 2175 2180 2185 GAG ACG GAC ATT GCA TCA TTC GAC AAA AGC CAG GAC GAC TCC TTG GCT 6689 Glu Thr Asp Ile Ala Ser Phe Asp Lys Ser Gln Asp Asp Ser Leu Ala 2190 2195 2200 CTT ACA GGT TTA ATG ATC CTC GAA GAT CTA GGG GTG GAT CAG TAC CTG 6737 Leu Thr Gly Leu Met Ile Leu Glu Asp Leu Gly Val Asp Gln Tyr Leu 2205 2210 2215 CTG GAC TTG ATC GAG GCA GCC TTT GGG GAA ATA TCC AGC TGT CAC CTA 6785 Leu Asp Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser Cys His Leu 2220 2225 2230 CCA ACT GGC ACG CGC TTC AAG TTC GGA GCT ATG ATG AAA TCG GGC ATG 6833 Pro Thr Gly Thr Arg Phe Lys Phe Gly Ala Met Met Lys Ser Gly Met 2235 2240 2245 TTT CTG ACT TTG TTT ATT AAC ACT GTT TTG AAC ATC ACC ATA GCA AGC 6881 Phe Leu Thr Leu Phe Ile Asn Thr Val Leu Asn Ile Thr Ile Ala Ser 2250 2255 2260 2265 AGG GTA CTG GAG CAG AGA CTC ACT GAC TCC GCC TGT GCG GCC TTC ATC 6929 Arg Val Leu Glu Gln Arg Leu Thr Asp Ser Ala Cys Ala Ala Phe Ile 2270 2275 2280 GGC GAC GAC AAC ATC GTT CAC GGA GTG ATC TCC GAC AAG CTG ATG GCG 6977 Gly Asp Asp Asn Ile Val His Gly Val Ile Ser Asp Lys Leu Met Ala 2285 2290 2295 GAG AGG TGC GCG TCG TGG GTC AAC ATG GAG GTG AAG ATC ATT GAC GCT 7025 Glu Arg Cys Ala Ser Trp Val Asn Met Glu Val Lys Ile Ile Asp Ala 2300 2305 2310 GTC ATG GGC GAA AAA CCC CCA TAT TTT TGT GGG GGA TTC ATA GTT TTT 7073 Val Met Gly Glu Lys Pro Pro Tyr Phe Cys Gly Gly Phe Ile Val Phe 2315 2320 2325 GAC AGC GTC ACA CAG ACC GCC TGC CGT GTT TCA GAC CCA CTT AAG CGC 7121 Asp Ser Val Thr Gln Thr Ala Cys Arg Val Ser Asp Pro Leu Lys Arg 2330 2335 2340 2345 CTG TTC AAG TTG GGT AAG CCG CTA ACA GCT GAA GAC AAG CAG GAC GAA 7169 Leu Phe Lys Leu Gly Lys Pro Leu Thr Ala Glu Asp Lys Gln Asp Glu 2350 2355 2360 GAC AGG CGA CGA GCA CTG AGT GAC GAG GTT AGC AAG TGG TTC CGG ACA 7217 Asp Arg Arg Arg Ala Leu Ser Asp Glu Val Ser Lys Trp Phe Arg Thr 2365 2370 2375 GGC TTG GGG GCC GAA CTG GAG GTG GCA CTA ACA TCT AGG TAT GAG GTA 7265 Gly Leu Gly Ala Glu Leu Glu Val Ala Leu Thr Ser Arg Tyr Glu Val 2380 2385 2390 GAG GGC TGC AAA AGT ATC CTC ATA GCC ATG ACC ACC TTG GCG AGG GAC 7313 Glu Gly Cys Lys Ser Ile Leu Ile Ala Met Thr Thr Leu Ala Arg Asp 2395 2400 2405 ATT AAG GCG TTT AAG AAA TTG AGA GGA CCT GTT ATA CAC CTC TAC GGC 7361 Ile Lys Ala Phe Lys Lys Leu Arg Gly Pro Val Ile His Leu Tyr Gly 2410 2415 2420 2425 GGT CCT AGA TTG GTG CGT TAATACACAG AATTCTGATT ATAGCGCACT 7409 Gly Pro Arg Leu Val Arg 2430 ATTATAGCAC C ATG AAT TAC ATC CCT ACG CAA ACG TTT TAC GGC CGC CGG 7459 Met Asn Tyr Ile Pro Thr Gln Thr Phe Tyr Gly Arg Arg 1 5 10 TGG CGC CCG CGC CCG GCG GCC CGT CCT TGG CCG TTG CAG GCC ACT CCG 7507 Trp Arg Pro Arg Pro Ala Ala Arg Pro Trp Pro Leu Gln Ala Thr Pro 15 20 25 GTG GCT CCC GTC GTC CCC GAC TTC CAG GCC CAG CAG ATG CAG CAA CTC 7555 Val Ala Pro Val Val Pro Asp Phe Gln Ala Gln Gln Met Gln Gln Leu 30 35 40 45 ATC AGC GCC GTA AAT GCG CTG ACA ATG AGA CAG AAC GCA ATT GCT CCT 7603 Ile Ser Ala Val Asn Ala Leu Thr Met Arg Gln Asn Ala Ile Ala Pro 50 55 60 GCT AGG CCT CCC AAA CCA AAG AAG AAG AAG ACA ACC AAA CCA AAG CCG 7651 Ala Arg Pro Pro Lys Pro Lys Lys Lys Lys Thr Thr Lys Pro Lys Pro 65 70 75 AAA ACG CAG CCC AAG AAG ATC AAC GGA AAA ACG CAG CAG CAA AAG AAG 7699 Lys Thr Gln Pro Lys Lys Ile Asn Gly Lys Thr Gln Gln Gln Lys Lys 80 85 90 AAA GAC AAG CAA GCC GAC AAG AAG AAG AAG AAA CCC GGA AAA AGA GAA 7747 Lys Asp Lys Gln Ala Asp Lys Lys Lys Lys Lys Pro Gly Lys Arg Glu 95 100 105 AGA ATG TGC ATG AAG ATT GAA AAT GAC TGT ATC TTC GAA GTC AAA CAC 7795 Arg Met Cys Met Lys Ile Glu Asn Asp Cys Ile Phe Glu Val Lys His 110 115 120 125 GAA GGA AAG GTC ACT GGG TAC GCC TGC CTG GTG GGC GAC AAA GTC ATG 7843 Glu Gly Lys Val Thr Gly Tyr Ala Cys Leu Val Gly Asp Lys Val Met 130 135 140 AAA CCT GCC CAC GTG AAA GGA GTC ATC GAC AAC GCG GAC CTG GCA AAG 7891 Lys Pro Ala His Val Lys Gly Val Ile Asp Asn Ala Asp Leu Ala Lys 145 150 155 CTA GCT TTC AAG AAA TCG AGC AAG TAT GAC CTT GAG TGT GCC CAG ATA 7939 Leu Ala Phe Lys Lys Ser Ser Lys Tyr Asp Leu Glu Cys Ala Gln Ile 160 165 170 CCA GTT CAC ATG AGG TCG GAT GCC TCA AAG TAC ACG CAT GAG AAG CCC 7987 Pro Val His Met Arg Ser Asp Ala Ser Lys Tyr Thr His Glu Lys Pro 175 180 185 GAG GGA CAC TAT AAC TGG CAC CAC GGG GCT GTT CAG TAC AGC GGA GGT 8035 Glu Gly His Tyr Asn Trp His His Gly Ala Val Gln Tyr Ser Gly Gly 190 195 200 205 AGG TTC ACT ATA CCG ACA GGA GCG GGC AAA CCG GGA GAC AGT GGC CGG 8083 Arg Phe Thr Ile Pro Thr Gly Ala Gly Lys Pro Gly Asp Ser Gly Arg 210 215 220 CCC ATC TTT GAC AAC AAG GGG AGG GTA GTC GCT ATC GTC CTG GGC GGG 8131 Pro Ile Phe Asp Asn Lys Gly Arg Val Val Ala Ile Val Leu Gly Gly 225 230 235 GCC AAC GAG GGC TCA CGC ACA GCA CTG TCG GTG GTC ACC TGG AAC AAA 8179 Ala Asn Glu Gly Ser Arg Thr Ala Leu Ser Val Val Thr Trp Asn Lys 240 245 250 GAT ATG GTG ACT AGA GTG ACC CCC GAG GGG TCC GAA GAG TGG TCC GCC 8227 Asp Met Val Thr Arg Val Thr Pro Glu Gly Ser Glu Glu Trp Ser Ala 255 260 265 CCG CTG ATT ACT GCC ATG TGT GTC CTT GCC AAT GCT ACC TTC CCG TGC 8275 Pro Leu Ile Thr Ala Met Cys Val Leu Ala Asn Ala Thr Phe Pro Cys 270 275 280 285 TTC CAG CCC CCG TGT GTA CCT TGC TGC TAT GAA AAC AAC GCA GAG GCC 8323 Phe Gln Pro Pro Cys Val Pro Cys Cys Tyr Glu Asn Asn Ala Glu Ala 290 295 300 ACA CTA CGG ATG CTC GAG GAT AAC GTG GAT AGG CCA GGG TAC TAC GAC 8371 Thr Leu Arg Met Leu Glu Asp Asn Val Asp Arg Pro Gly Tyr Tyr Asp 305 310 315 CTC CTT CAG GCA GCC TTG ACG TGC CGA AAC GGA ACA AGA CAC CGG CGC 8419 Leu Leu Gln Ala Ala Leu Thr Cys Arg Asn Gly Thr Arg His Arg Arg 320 325 330 AGC GTG TCG CAA CAC TTC AAC GTG TAT AAG GCT ACA CGC CCT TAC ATC 8467 Ser Val Ser Gln His Phe Asn Val Tyr Lys Ala Thr Arg Pro Tyr Ile 335 340 345 GCG TAC TGC GCC GAC TGC GGA GCA GGG CAC TCG TGT CAT AGC CCC GTA 8515 Ala Tyr Cys Ala Asp Cys Gly Ala Gly His Ser Cys His Ser Pro Val 350 355 360 365 GCA ATT GAA GCG GTC AGG TCC GAA GCT ACC GAC GGG ATG CTG AAG ATT 8563 Ala Ile Glu Ala Val Arg Ser Glu Ala Thr Asp Gly Met Leu Lys Ile 370 375 380 CAG TTC TCG GCA CAA ATT GGC ATA GAT AAG AGT GAC AAT CAT GAC TAC 8611 Gln Phe Ser Ala Gln Ile Gly Ile Asp Lys Ser Asp Asn His Asp Tyr 385 390 395 ACG AAG ATA AGG TAC GCA GAC GGG CAC GCC ATT GAG AAT GCC GTC CGG 8659 Thr Lys Ile Arg Tyr Ala Asp Gly His Ala Ile Glu Asn Ala Val Arg 400 405 410 TCA TCT TTG AAG GTA GCC ACC TCC GGA GAC TGT TTC GTC CAT GGC ACA 8707 Ser Ser Leu Lys Val Ala Thr Ser Gly Asp Cys Phe Val His Gly Thr 415 420 425 ATG GGA CAT TTC ATA CTG GCA AAG TGC CCA CCG GGT GAA TTC CTG CAG 8755 Met Gly His Phe Ile Leu Ala Lys Cys Pro Pro Gly Glu Phe Leu Gln 430 435 440 445 GTC TCG ATC CAG GAC ACC AGA AAC GCG GTC CGT GCC TGC AGA ATA CAA 8803 Val Ser Ile Gln Asp Thr Arg Asn Ala Val Arg Ala Cys Arg Ile Gln 450 455 460 TAT CAT CAT GAC CCT CAA CCG GTG GGT AGA GAA AAA TTT ACA ATT AGA 8851 Tyr His His Asp Pro Gln Pro Val Gly Arg Glu Lys Phe Thr Ile Arg 465 470 475 CCA CAC TAT GGA AAA GAG ATC CCT TGC ACC ACT TAT CAA CAG ACC ACA 8899 Pro His Tyr Gly Lys Glu Ile Pro Cys Thr Thr Tyr Gln Gln Thr Thr 480 485 490 GCG AAG ACC GTG GAG GAA ATC GAC ATG CAT ATG CCG CCA GAT ACG CCG 8947 Ala Lys Thr Val Glu Glu Ile Asp Met His Met Pro Pro Asp Thr Pro 495 500 505 GAC AGG ACG TTG CTA TCA CAG CAA TCT GGC AAT GTA AAG ATC ACA GTC 8995 Asp Arg Thr Leu Leu Ser Gln Gln Ser Gly Asn Val Lys Ile Thr Val 510 515 520 525 GGA GGA AAG AAG GTG AAA TAC AAC TGC ACC TGT GGA ACC GGA AAC GTT 9043 Gly Gly Lys Lys Val Lys Tyr Asn Cys Thr Cys Gly Thr Gly Asn Val 530 535 540 GGC ACT ACT AAT TCG GAC ATG ACG ATC AAC ACG TGT CTA ATA GAG CAG 9091 Gly Thr Thr Asn Ser Asp Met Thr Ile Asn Thr Cys Leu Ile Glu Gln 545 550 555 TGC CAC GTC TCA GTG ACG GAC CAT AAG AAA TGG CAG TTC AAC TCA CCT 9139 Cys His Val Ser Val Thr Asp His Lys Lys Trp Gln Phe Asn Ser Pro 560 565 570 TTC GTC CCG AGA GCC GAC GAA CCG GCT AGA AAA GGC AAA GTC CAT ATC 9187 Phe Val Pro Arg Ala Asp Glu Pro Ala Arg Lys Gly Lys Val His Ile 575 580 585 CCA TTC CCG TTG GAC AAC ATC ACA TGC AGA GTT CCA ATG GCG CGC GAA 9235 Pro Phe Pro Leu Asp Asn Ile Thr Cys Arg Val Pro Met Ala Arg Glu 590 595 600 605 CCA ACC GTC ATC CAC GGC AAA AGA GAA GTG ACA CTG CAC CTT CAC CCA 9283 Pro Thr Val Ile His Gly Lys Arg Glu Val Thr Leu His Leu His Pro 610 615 620 GAT CAT CCC ACG CTC TTT TCC TAC CGC ACA CTG GGT GAG GAC CCG CAG 9331 Asp His Pro Thr Leu Phe Ser Tyr Arg Thr Leu Gly Glu Asp Pro Gln 625 630 635 TAT CAC GAG GAA TGG GTG ACA GCG GCG GTG GAA CGG ACC ATA CCC GTA 9379 Tyr His Glu Glu Trp Val Thr Ala Ala Val Glu Arg Thr Ile Pro Val 640 645 650 CCA GTG GAC GGG ATG GAG TAC CAC TGG GGA AAC AAC GAC CCA GTG AGG 9427 Pro Val Asp Gly Met Glu Tyr His Trp Gly Asn Asn Asp Pro Val Arg 655 660 665 CTT TGG TCT CAA CTC ACC ACT GAA GGG AAA CCG CAC GGC TGG CCG CAT 9475 Leu Trp Ser Gln Leu Thr Thr Glu Gly Lys Pro His Gly Trp Pro His 670 675 680 685 CAG ATC GTA CAG TAC TAC TAT GGG CTT TAC CCG GCC GCT ACA GTA TCC 9523 Gln Ile Val Gln Tyr Tyr Tyr Gly Leu Tyr Pro Ala Ala Thr Val Ser 690 695 700 GCG GTC GTC GGG ATG AGC TTA CTG GCG TTG ATA TCG ATC TTC GCG TCG 9571 Ala Val Val Gly Met Ser Leu Leu Ala Leu Ile Ser Ile Phe Ala Ser 705 710 715 TGC TAC ATG CTG GTT GCG GCC CGC AGT AAG TGC TTG ACC CCT TAT GCT 9619 Cys Tyr Met Leu Val Ala Ala Arg Ser Lys Cys Leu Thr Pro Tyr Ala 720 725 730 TTA ACA CCA GGA GCT GCA GTT CCG TGG ACG CTG GGG ATA CTC TGC TGC 9667 Leu Thr Pro Gly Ala Ala Val Pro Trp Thr Leu Gly Ile Leu Cys Cys 735 740 745 GCC CCG CGG GCG CAC GCA GCT AGT GTG GCA GAG ACT ATG GCC TAC TTG 9715 Ala Pro Arg Ala His Ala Ala Ser Val Ala Glu Thr Met Ala Tyr Leu 750 755 760 765 TGG GAC CAA AAC CAA GCG TTG TTC TGG TTG GAG TTT GCG GCC CCT GTT 9763 Trp Asp Gln Asn Gln Ala Leu Phe Trp Leu Glu Phe Ala Ala Pro Val 770 775 780 GCC TGC ATC CTC ATC ATC ACG TAT TGC CTC AGA AAC GTG CTG TGT TGC 9811 Ala Cys Ile Leu Ile Ile Thr Tyr Cys Leu Arg Asn Val Leu Cys Cys 785 790 795 TGT AAG AGC CTT TCT TTT TTA GTG CTA CTG AGC CTC GGG GCA ACC GCC 9859 Cys Lys Ser Leu Ser Phe Leu Val Leu Leu Ser Leu Gly Ala Thr Ala 800 805 810 AGA GCT TAC GAA CAT TCG ACA GTA ATG CCG AAC GTG GTG GGG TTC CCG 9907 Arg Ala Tyr Glu His Ser Thr Val Met Pro Asn Val Val Gly Phe Pro 815 820 825 TAT AAG GCT CAC ATT GAA AGG CCA GGA TAT AGC CCC CTC ACT TTG CAG 9955 Tyr Lys Ala His Ile Glu Arg Pro Gly Tyr Ser Pro Leu Thr Leu Gln 830 835 840 845 ATG CAG GTT GTT GAA ACC AGC CTC GAA CCA ACC CTT AAT TTG GAA TAC 10003 Met Gln Val Val Glu Thr Ser Leu Glu Pro Thr Leu Asn Leu Glu Tyr 850 855 860 ATA ACC TGT GAG TAC AAG ACG GTC GTC CCG TCG CCG TAC GTG AAG TGC 10051 Ile Thr Cys Glu Tyr Lys Thr Val Val Pro Ser Pro Tyr Val Lys Cys 865 870 875 TGC GGC GCC TCA GAG TGC TCC ACT AAA GAG AAG CCT GAC TAC CAA TGC 10099 Cys Gly Ala Ser Glu Cys Ser Thr Lys Glu Lys Pro Asp Tyr Gln Cys 880 885 890 AAG GTT TAC ACA GGC GTG TAC CCG TTC ATG TGG GGA GGG GCA TAT TGC 10147 Lys Val Tyr Thr Gly Val Tyr Pro Phe Met Trp Gly Gly Ala Tyr Cys 895 900 905 TTC TGC GAC TCA GAA AAC ACG CAA CTC AGC GAG GCG TAC GTC GAT CGA 10195 Phe Cys Asp Ser Glu Asn Thr Gln Leu Ser Glu Ala Tyr Val Asp Arg 910 915 920 925 TCG GAC GTA TGC AGG CAT GAT CAC GCA TCT GCT TAC AAA GCC CAT ACA 10243 Ser Asp Val Cys Arg His Asp His Ala Ser Ala Tyr Lys Ala His Thr 930 935 940 GCA TCG CTG AAG GCC AAA GTG AGG GTT ATG TAC GGC AAC GTA AAC CAG 10291 Ala Ser Leu Lys Ala Lys Val Arg Val Met Tyr Gly Asn Val Asn Gln 945 950 955 ACT GTG GAT GTT TAC GTG AAC GGA GAC CAT GCC GTC ACG ATA GGG GGT 10339 Thr Val Asp Val Tyr Val Asn Gly Asp His Ala Val Thr Ile Gly Gly 960 965 970 ACT CAG TTC ATA TTC GGG CCG CTG TCA TCG GCC TGG ACC CCG TTC GAC 10387 Thr Gln Phe Ile Phe Gly Pro Leu Ser Ser Ala Trp Thr Pro Phe Asp 975 980 985 AAC AAG ATA GTC GTG TAC AAA GAC GAA GTG TTC AAT CAG GAC TTC CCG 10435 Asn Lys Ile Val Val Tyr Lys Asp Glu Val Phe Asn Gln Asp Phe Pro 990 995 1000 1005 CCG TAC GGA TCT GGG CAA CCA GGG CGC TTC GGC GAC ATC CAA AGC AGA 10483 Pro Tyr Gly Ser Gly Gln Pro Gly Arg Phe Gly Asp Ile Gln Ser Arg 1010 1015 1020 ACA GTG GAG AGT AAC GAC CTG TAC GCG AAC ACG GCA CTG AAG CTG GCA 10531 Thr Val Glu Ser Asn Asp Leu Tyr Ala Asn Thr Ala Leu Lys Leu Ala 1025 1030 1035 CGC CCT TCA CCC GGC ATG GTC CAT GTA CCG TAC ACA CAG ACA CCT TCA 10579 Arg Pro Ser Pro Gly Met Val His Val Pro Tyr Thr Gln Thr Pro Ser 1040 1045 1050 GGG TTC AAA TAT TGG CTA AAG GAA AAA GGG ACA GCC CTA AAT ACG AAG 10627 Gly Phe Lys Tyr Trp Leu Lys Glu Lys Gly Thr Ala Leu Asn Thr Lys 1055 1060 1065 GCT CCT TTT GGC TGC CAA ATC AAA ACG AAC CCT GTC AGG GCC ATG AAC 10675 Ala Pro Phe Gly Cys Gln Ile Lys Thr Asn Pro Val Arg Ala Met Asn 1070 1075 1080 1085 TGC GCC GTG GGA AAC ATC CCT GTC TCC ATG AAT TTG CCT GAC AGC GCC 10723 Cys Ala Val Gly Asn Ile Pro Val Ser Met Asn Leu Pro Asp Ser Ala 1090 1095 1100 TTT ACC CGC ATT GTC GAG GCG CCG ACC ATC ATT GAC CTG ACT TGC ACA 10771 Phe Thr Arg Ile Val Glu Ala Pro Thr Ile Ile Asp Leu Thr Cys Thr 1105 1110 1115 GTG GCT ACC TGT ACG CAC TCC TCG GAT TTC GGC GGC GTC TTG ACA CTG 10819 Val Ala Thr Cys Thr His Ser Ser Asp Phe Gly Gly Val Leu Thr Leu 1120 1125 1130 ACG TAC AAG ACC AAC AAG AAC GGG GAC TGC TCT GTA CAC TCG CAC TCT 10867 Thr Tyr Lys Thr Asn Lys Asn Gly Asp Cys Ser Val His Ser His Ser 1135 1140 1145 AAC GTA GCT ACT CTA CAG GAG GCC ACA GCA AAA GTG AAG ACA GCA GGT 10915 Asn Val Ala Thr Leu Gln Glu Ala Thr Ala Lys Val Lys Thr Ala Gly 1150 1155 1160 1165 AAG GTG ACC TTA CAC TTC TCC ACG GCA AGC GCA TCA CCT TCT TTT GTG 10963 Lys Val Thr Leu His Phe Ser Thr Ala Ser Ala Ser Pro Ser Phe Val 1170 1175 1180 GTG TCG CTA TGC AGT GCT AGG GCC ACC TGT TCA GCG TCG TGT GAG CCC 11011 Val Ser Leu Cys Ser Ala Arg Ala Thr Cys Ser Ala Ser Cys Glu Pro 1185 1190 1195 CCG AAA GAC CAC ATA GTC CCA TAT GCG GCT AGC CAC AGT AAC GTA GTG 11059 Pro Lys Asp His Ile Val Pro Tyr Ala Ala Ser His Ser Asn Val Val 1200 1205 1210 TTT CCA GAC ATG TCG GGC ACC GCA CTA TCA TGG GTG CAG AAA ATC TCG 11107 Phe Pro Asp Met Ser Gly Thr Ala Leu Ser Trp Val Gln Lys Ile Ser 1215 1220 1225 GGT GGT CTG GGG GCC TTC GCA ATC GGC GCT ATC CTG GTG CTG GTT GTG 11155 Gly Gly Leu Gly Ala Phe Ala Ile Gly Ala Ile Leu Val Leu Val Val 1230 1235 1240 1245 GTC ACT TGC ATT GGG CTC CGC AGA TAAGTTAGGG TAGGCAATGG CATTGATATA 11209 Val Thr Cys Ile Gly Leu Arg Arg 1250 GCAAGAAAAT TGAAAACAGA AAAAGTTAGG GTAAGCAATG GCATATAACC ATAACTGTAT 11269 AACTTGTAAC AAAGCGCAAC AAGACCTGCG CAATTGGCCC CGTGGTCCGC CTCACGGAAA 11329 CTCGGGGCAA CTCATATTGA CACATTAATT GGCAATAATT GGAAGCTTAC ATAAGCTTAA 11389 TTCGACGAAT AATTGGATTT TTATTTTATT TTGCAATTGG TTTTTAATAT TTCCAAAAAA 11449 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 11509 AAAACTAG 11517 2431 amino acids amino acid linear protein 2 Met Ala Ala Lys Val His Val Asp Ile Glu Ala Asp Ser Pro Phe Ile 1 5 10 15 Lys Ser Leu Gln Lys Ala Phe Pro Ser Phe Glu Val Glu Ser Leu Gln 20 25 30 Val Thr Pro Asn Asp His Ala Asn Ala Arg Ala Phe Ser His Leu Ala 35 40 45 Thr Lys Leu Ile Glu Gln Glu Thr Asp Lys Asp Thr Leu Ile Leu Asp 50 55 60 Ile Gly Ser Ala Pro Ser Arg Arg Met Met Ser Thr His Lys Tyr His 65 70 75 80 Cys Val Cys Pro Met Arg Ser Ala Glu Asp Pro Glu Arg Leu Asp Ser 85 90 95 Tyr Ala Lys Lys Leu Ala Ala Ala Ser Gly Lys Val Leu Asp Arg Glu 100 105 110 Ile Ala Gly Lys Ile Thr Asp Leu Gln Thr Val Met Ala Thr Pro Asp 115 120 125 Ala Glu Ser Pro Thr Phe Cys Leu His Thr Asp Val Thr Cys Arg Thr 130 135 140 Ala Ala Glu Val Ala Val Tyr Gln Asp Val Tyr Ala Val His Ala Pro 145 150 155 160 Thr Ser Leu Tyr His Gln Ala Met Lys Gly Val Arg Thr Ala Tyr Trp 165 170 175 Ile Gly Phe Asp Thr Thr Pro Phe Met Phe Asp Ala Leu Ala Gly Ala 180 185 190 Tyr Pro Thr Tyr Ala Thr Asn Trp Ala Asp Glu Gln Val Leu Gln Ala 195 200 205 Arg Asn Ile Gly Leu Cys Ala Ala Ser Leu Thr Glu Gly Arg Leu Gly 210 215 220 Lys Leu Ser Ile Leu Arg Lys Lys Gln Leu Lys Pro Cys Asp Thr Val 225 230 235 240 Met Phe Ser Val Gly Ser Thr Leu Tyr Thr Glu Ser Arg Lys Leu Leu 245 250 255 Arg Ser Trp His Leu Pro Ser Val Phe His Leu Lys Gly Lys Gln Ser 260 265 270 Phe Thr Cys Arg Cys Asp Thr Ile Val Ser Cys Glu Gly Tyr Val Val 275 280 285 Lys Lys Ile Thr Met Cys Pro Gly Leu Tyr Gly Lys Thr Val Gly Tyr 290 295 300 Ala Val Thr Tyr His Ala Glu Gly Phe Leu Val Cys Lys Thr Thr Asp 305 310 315 320 Thr Val Lys Gly Glu Arg Val Ser Phe Pro Val Cys Thr Tyr Val Pro 325 330 335 Ser Thr Ile Cys Asp Gln Met Thr Gly Ile Leu Ala Thr Asp Val Thr 340 345 350 Pro Glu Asp Ala Gln Lys Leu Leu Val Gly Leu Asn Gln Arg Ile Val 355 360 365 Val Asn Gly Arg Thr Gln Arg Asn Thr Asn Thr Met Lys Asn Tyr Leu 370 375 380 Leu Pro Ile Val Ala Val Ala Phe Ser Lys Trp Ala Arg Glu Tyr Lys 385 390 395 400 Ala Asp Leu Asp Asp Glu Lys Pro Leu Gly Val Arg Glu Arg Ser Leu 405 410 415 Thr Cys Cys Cys Leu Trp Ala Phe Lys Thr Arg Lys Met His Thr Met 420 425 430 Tyr Lys Lys Pro Asp Thr Gln Thr Ile Val Lys Val Pro Ser Glu Phe 435 440 445 Asn Ser Phe Val Ile Pro Ser Leu Trp Ser Thr Gly Leu Ala Ile Pro 450 455 460 Val Arg Ser Arg Ile Lys Met Leu Leu Ala Lys Lys Thr Lys Arg Glu 465 470 475 480 Leu Ile Pro Val Leu Asp Ala Ser Ser Ala Arg Asp Ala Glu Gln Glu 485 490 495 Glu Lys Glu Arg Leu Glu Ala Glu Leu Thr Arg Glu Ala Leu Pro Pro 500 505 510 Leu Val Pro Ile Ala Pro Ala Glu Thr Gly Val Val Asp Val Asp Val 515 520 525 Glu Glu Leu Glu Tyr His Ala Gly Ala Gly Val Val Glu Thr Pro Arg 530 535 540 Ser Ala Leu Lys Val Thr Ala Gln Pro Asn Asp Val Leu Leu Gly Asn 545 550 555 560 Tyr Val Val Leu Ser Pro Gln Thr Val Leu Lys Ser Ser Lys Leu Ala 565 570 575 Pro Val His Pro Leu Ala Glu Gln Val Lys Ile Ile Thr His Asn Gly 580 585 590 Arg Ala Gly Gly Tyr Gln Val Asp Gly Tyr Asp Gly Arg Val Leu Leu 595 600 605 Pro Cys Gly Ser Ala Ile Pro Val Pro Glu Phe Gln Ala Leu Ser Glu 610 615 620 Ser Ala Thr Met Val Tyr Asn Glu Arg Glu Phe Val Asn Arg Lys Leu 625 630 635 640 Tyr His Ile Ala Val His Gly Pro Ser Leu Asn Thr Asp Glu Glu Asn 645 650 655 Tyr Glu Lys Val Arg Ala Glu Arg Thr Asp Ala Glu Tyr Val Phe Asp 660 665 670 Val Asp Lys Lys Cys Cys Val Lys Arg Glu Glu Ala Ser Gly Leu Val 675 680 685 Leu Val Gly Glu Leu Thr Asn Pro Pro Phe His Glu Phe Ala Tyr Glu 690 695 700 Gly Leu Lys Ile Arg Pro Ser Ala Pro Tyr Lys Thr Thr Val Val Gly 705 710 715 720 Val Phe Gly Val Pro Gly Ser Gly Lys Ser Ala Ile Ile Lys Ser Leu 725 730 735 Val Thr Lys His Asp Leu Val Thr Ser Gly Lys Lys Glu Asn Cys Gln 740 745 750 Glu Ile Val Asn Asp Val Lys Lys His Arg Gly Lys Gly Thr Ser Arg 755 760 765 Glu Asn Ser Asp Ser Ile Leu Leu Asn Gly Cys Arg Arg Ala Val Asp 770 775 780 Ile Leu Tyr Val Asp Glu Ala Phe Ala Cys His Ser Gly Thr Leu Leu 785 790 795 800 Ala Leu Ile Ala Leu Val Lys Pro Arg Ser Lys Val Val Leu Cys Gly 805 810 815 Asp Pro Lys Gln Cys Gly Phe Phe Asn Met Met Gln Leu Lys Val Asn 820 825 830 Phe Asn His Asn Ile Cys Thr Glu Val Cys His Lys Ser Ile Ser Arg 835 840 845 Arg Cys Thr Arg Pro Val Thr Ala Ile Val Ser Thr Leu His Tyr Gly 850 855 860 Gly Lys Met Arg Thr Thr Asn Pro Cys Asn Lys Pro Ile Ile Ile Asp 865 870 875 880 Thr Thr Gly Gln Thr Lys Pro Lys Pro Gly Asp Ile Val Leu Thr Cys 885 890 895 Phe Arg Gly Trp Ala Lys Gln Leu Gln Leu Asp Tyr Arg Gly His Glu 900 905 910 Val Met Thr Ala Ala Ala Ser Gln Gly Leu Thr Arg Lys Gly Val Tyr 915 920 925 Ala Val Arg Gln Lys Val Asn Glu Asn Pro Leu Tyr Ala Pro Ala Ser 930 935 940 Glu His Val Asn Val Leu Leu Thr Arg Thr Glu Asp Arg Leu Val Trp 945 950 955 960 Lys Thr Leu Ala Gly Asp Pro Trp Ile Lys Val Leu Ser Asn Ile Pro 965 970 975 Gln Gly Asn Phe Thr Ala Thr Leu Glu Glu Trp Gln Glu Glu His Asp 980 985 990 Lys Ile Met Lys Val Ile Glu Gly Pro Ala Ala Pro Val Asp Ala Phe 995 1000 1005 Gln Asn Lys Ala Asn Val Cys Trp Ala Lys Ser Leu Val Pro Val Leu 1010 1015 1020 Asp Thr Ala Gly Ile Arg Leu Thr Ala Glu Glu Trp Ser Thr Ile Ile 1025 1030 1035 1040 Thr Ala Phe Lys Glu Asp Arg Ala Tyr Ser Pro Val Val Ala Leu Asn 1045 1050 1055 Glu Ile Cys Thr Lys Tyr Tyr Gly Val Asp Leu Asp Ser Gly Leu Phe 1060 1065 1070 Ser Ala Pro Lys Val Ser Leu Tyr Tyr Glu Asn Asn His Trp Asp Asn 1075 1080 1085 Arg Pro Gly Gly Arg Met Tyr Gly Phe Asn Ala Ala Thr Ala Ala Arg 1090 1095 1100 Leu Glu Ala Arg His Thr Phe Leu Lys Gly Gln Trp His Thr Gly Lys 1105 1110 1115 1120 Gln Ala Val Ile Ala Glu Arg Lys Ile Gln Pro Leu Ser Val Leu Asp 1125 1130 1135 Asn Val Ile Pro Ile Asn Arg Arg Leu Pro His Ala Leu Val Ala Glu 1140 1145 1150 Tyr Lys Thr Val Lys Gly Ser Arg Val Glu Trp Leu Val Asn Lys Val 1155 1160 1165 Arg Gly Tyr His Val Leu Leu Val Ser Glu Tyr Asn Leu Ala Leu Pro 1170 1175 1180 Arg Arg Arg Val Thr Trp Leu Ser Pro Leu Asn Val Thr Gly Ala Asp 1185 1190 1195 1200 Arg Cys Tyr Asp Leu Ser Leu Gly Leu Pro Ala Asp Ala Gly Arg Phe 1205 1210 1215 Asp Leu Val Phe Val Asn Ile His Thr Glu Phe Arg Ile His His Tyr 1220 1225 1230 Gln Gln Cys Val Asp His Ala Met Lys Leu Gln Met Leu Gly Gly Asp 1235 1240 1245 Ala Leu Arg Leu Leu Lys Pro Gly Gly Ile Leu Met Arg Ala Tyr Gly 1250 1255 1260 Tyr Ala Asp Lys Ile Ser Glu Ala Val Val Ser Ser Leu Ser Arg Lys 1265 1270 1275 1280 Phe Ser Ser Ala Arg Val Leu Arg Pro Asp Cys Val Thr Ser Asn Thr 1285 1290 1295 Glu Val Phe Leu Leu Phe Ser Asn Phe Asp Asn Gly Lys Arg Pro Ser 1300 1305 1310 Thr Leu His Gln Met Asn Thr Lys Leu Ser Ala Val Tyr Ala Gly Glu 1315 1320 1325 Ala Met His Thr Ala Gly Cys Ala Pro Ser Tyr Arg Val Lys Arg Ala 1330 1335 1340 Asp Ile Ala Thr Cys Thr Glu Ala Ala Val Val Asn Ala Ala Asn Ala 1345 1350 1355 1360 Arg Gly Thr Val Gly Asp Gly Val Cys Arg Ala Val Ala Lys Lys Trp 1365 1370 1375 Pro Ser Ala Phe Lys Gly Ala Ala Thr Pro Val Gly Thr Ile Lys Thr 1380 1385 1390 Val Met Cys Gly Ser Tyr Pro Val Ile His Ala Val Ala Pro Asn Phe 1395 1400 1405 Ser Ala Thr Thr Glu Ala Glu Gly Asp Arg Glu Leu Ala Ala Val Tyr 1410 1415 1420 Arg Ala Val Ala Ala Glu Val Asn Arg Leu Ser Leu Ser Ser Val Ala 1425 1430 1435 1440 Ile Pro Leu Leu Ser Thr Gly Val Phe Ser Gly Gly Arg Asp Arg Leu 1445 1450 1455 Gln Gln Ser Leu Asn His Leu Phe Thr Ala Met Asp Ala Thr Asp Ala 1460 1465 1470 Asp Val Thr Ile Tyr Cys Arg Asp Lys Ser Trp Glu Lys Lys Ile Gln 1475 1480 1485 Glu Ala Ile Asp Met Arg Thr Ala Val Glu Leu Leu Asn Asp Asp Val 1490 1495 1500 Glu Leu Thr Thr Asp Leu Val Arg Val His Pro Asp Ser Ser Leu Val 1505 1510 1515 1520 Gly Arg Lys Gly Tyr Ser Thr Thr Asp Gly Ser Leu Tyr Ser Tyr Phe 1525 1530 1535 Glu Gly Thr Lys Phe Asn Gln Ala Ala Ile Asp Met Ala Glu Ile Leu 1540 1545 1550 Thr Leu Trp Pro Arg Leu Gln Glu Ala Asn Glu Gln Ile Cys Leu Tyr 1555 1560 1565 Ala Leu Gly Glu Thr Met Asp Asn Ile Arg Ser Lys Cys Pro Val Asn 1570 1575 1580 Asp Ser Asp Ser Ser Thr Pro Pro Arg Thr Val Pro Cys Leu Cys Arg 1585 1590 1595 1600 Tyr Ala Met Thr Ala Glu Arg Ile Ala Arg Leu Arg Ser His Gln Val 1605 1610 1615 Lys Ser Met Val Val Cys Ser Ser Phe Pro Leu Pro Lys Tyr His Val 1620 1625 1630 Asp Gly Val Gln Lys Val Lys Cys Glu Lys Val Leu Leu Phe Asp Pro 1635 1640 1645 Thr Val Pro Ser Val Val Ser Pro Arg Lys Tyr Ala Ala Ser Thr Thr 1650 1655 1660 Asp His Ser Asp Arg Ser Leu Arg Gly Phe Asp Leu Asp Trp Thr Thr 1665 1670 1675 1680 Asp Ser Ser Ser Thr Ala Ser Asp Thr Met Ser Leu Pro Ser Leu Gln 1685 1690 1695 Ser Cys Asp Ile Asp Ser Ile Tyr Glu Pro Met Ala Pro Ile Val Val 1700 1705 1710 Thr Ala Asp Val His Pro Glu Pro Ala Gly Ile Ala Asp Leu Ala Ala 1715 1720 1725 Asp Val His Pro Glu Pro Ala Asp His Val Asp Leu Glu Asn Pro Ile 1730 1735 1740 Pro Pro Pro Arg Pro Lys Arg Ala Ala Tyr Leu Ala Ser Arg Ala Ala 1745 1750 1755 1760 Glu Arg Pro Val Pro Ala Pro Arg Lys Pro Thr Pro Ala Pro Arg Thr 1765 1770 1775 Ala Phe Arg Asn Lys Leu Pro Leu Thr Phe Gly Asp Phe Asp Glu His 1780 1785 1790 Glu Val Asp Ala Leu Ala Ser Gly Ile Thr Phe Gly Asp Phe Asp Asp 1795 1800 1805 Val Leu Arg Leu Gly Arg Ala Gly Ala Tyr Ile Phe Ser Ser Asp Thr 1810 1815 1820 Gly Ser Gly His Leu Gln Gln Lys Ser Val Arg Gln His Asn Leu Gln 1825 1830 1835 1840 Cys Ala Gln Leu Asp Ala Val Gln Glu Glu Lys Met Tyr Pro Pro Lys 1845 1850 1855 Leu Asp Thr Glu Arg Glu Lys Leu Leu Leu Leu Lys Met Gln Met His 1860 1865 1870 Pro Ser Glu Ala Asn Lys Ser Arg Tyr Gln Ser Arg Lys Val Glu Asn 1875 1880 1885 Met Lys Ala Thr Val Val Asp Arg Leu Thr Ser Gly Ala Arg Leu Tyr 1890 1895 1900 Thr Gly Ala Asp Val Gly Arg Ile Pro Thr Tyr Ala Val Arg Tyr Pro 1905 1910 1915 1920 Arg Pro Val Tyr Ser Pro Thr Val Ile Glu Arg Phe Ser Ser Pro Asp 1925 1930 1935 Val Ala Ile Ala Ala Cys Asn Glu Tyr Leu Ser Arg Asn Tyr Pro Thr 1940 1945 1950 Val Ala Ser Tyr Gln Ile Thr Asp Glu Tyr Asp Ala Tyr Leu Asp Met 1955 1960 1965 Val Asp Gly Ser Asp Ser Cys Leu Asp Arg Ala Thr Phe Cys Pro Ala 1970 1975 1980 Lys Leu Arg Cys Tyr Pro Lys His His Ala Tyr His Gln Pro Thr Val 1985 1990 1995 2000 Arg Ser Ala Val Pro Ser Pro Phe Gln Asn Thr Leu Gln Asn Val Leu 2005 2010 2015 Ala Ala Ala Thr Lys Arg Asn Cys Asn Val Thr Gln Met Arg Glu Leu 2020 2025 2030 Pro Thr Met Asp Ser Ala Val Phe Asn Val Glu Cys Phe Lys Arg Tyr 2035 2040 2045 Ala Cys Ser Gly Glu Tyr Trp Glu Glu Tyr Ala Lys Gln Pro Ile Arg 2050 2055 2060 Ile Thr Thr Glu Asn Ile Thr Thr Tyr Val Thr Lys Leu Lys Gly Pro 2065 2070 2075 2080 Lys Ala Ala Ala Leu Phe Ala Lys Thr His Asn Leu Val Pro Leu Gln 2085 2090 2095 Glu Val Pro Met Asp Arg Phe Thr Val Asp Met Lys Arg Asp Val Lys 2100 2105 2110 Val Thr Pro Gly Thr Lys His Thr Glu Glu Arg Pro Lys Val Gln Val 2115 2120 2125 Ile Gln Ala Ala Glu Pro Leu Ala Thr Ala Tyr Leu Cys Gly Ile His 2130 2135 2140 Arg Glu Leu Val Arg Arg Leu Asn Ala Val Leu Arg Pro Asn Val His 2145 2150 2155 2160 Thr Leu Phe Asp Met Ser Ala Glu Asp Phe Asp Ala Ile Ile Ala Ser 2165 2170 2175 His Phe His Pro Gly Asp Pro Val Leu Glu Thr Asp Ile Ala Ser Phe 2180 2185 2190 Asp Lys Ser Gln Asp Asp Ser Leu Ala Leu Thr Gly Leu Met Ile Leu 2195 2200 2205 Glu Asp Leu Gly Val Asp Gln Tyr Leu Leu Asp Leu Ile Glu Ala Ala 2210 2215 2220 Phe Gly Glu Ile Ser Ser Cys His Leu Pro Thr Gly Thr Arg Phe Lys 2225 2230 2235 2240 Phe Gly Ala Met Met Lys Ser Gly Met Phe Leu Thr Leu Phe Ile Asn 2245 2250 2255 Thr Val Leu Asn Ile Thr Ile Ala Ser Arg Val Leu Glu Gln Arg Leu 2260 2265 2270 Thr Asp Ser Ala Cys Ala Ala Phe Ile Gly Asp Asp Asn Ile Val His 2275 2280 2285 Gly Val Ile Ser Asp Lys Leu Met Ala Glu Arg Cys Ala Ser Trp Val 2290 2295 2300 Asn Met Glu Val Lys Ile Ile Asp Ala Val Met Gly Glu Lys Pro Pro 2305 2310 2315 2320 Tyr Phe Cys Gly Gly Phe Ile Val Phe Asp Ser Val Thr Gln Thr Ala 2325 2330 2335 Cys Arg Val Ser Asp Pro Leu Lys Arg Leu Phe Lys Leu Gly Lys Pro 2340 2345 2350 Leu Thr Ala Glu Asp Lys Gln Asp Glu Asp Arg Arg Arg Ala Leu Ser 2355 2360 2365 Asp Glu Val Ser Lys Trp Phe Arg Thr Gly Leu Gly Ala Glu Leu Glu 2370 2375 2380 Val Ala Leu Thr Ser Arg Tyr Glu Val Glu Gly Cys Lys Ser Ile Leu 2385 2390 2395 2400 Ile Ala Met Thr Thr Leu Ala Arg Asp Ile Lys Ala Phe Lys Lys Leu 2405 2410 2415 Arg Gly Pro Val Ile His Leu Tyr Gly Gly Pro Arg Leu Val Arg 2420 2425 2430 1253 amino acids amino acid linear protein 3 Met Asn Tyr Ile Pro Thr Gln Thr Phe Tyr Gly Arg Arg Trp Arg Pro 1 5 10 15 Arg Pro Ala Ala Arg Pro Trp Pro Leu Gln Ala Thr Pro Val Ala Pro 20 25 30 Val Val Pro Asp Phe Gln Ala Gln Gln Met Gln Gln Leu Ile Ser Ala 35 40 45 Val Asn Ala Leu Thr Met Arg Gln Asn Ala Ile Ala Pro Ala Arg Pro 50 55 60 Pro Lys Pro Lys Lys Lys Lys Thr Thr Lys Pro Lys Pro Lys Thr Gln 65 70 75 80 Pro Lys Lys Ile Asn Gly Lys Thr Gln Gln Gln Lys Lys Lys Asp Lys 85 90 95 Gln Ala Asp Lys Lys Lys Lys Lys Pro Gly Lys Arg Glu Arg Met Cys 100 105 110 Met Lys Ile Glu Asn Asp Cys Ile Phe Glu Val Lys His Glu Gly Lys 115 120 125 Val Thr Gly Tyr Ala Cys Leu Val Gly Asp Lys Val Met Lys Pro Ala 130 135 140 His Val Lys Gly Val Ile Asp Asn Ala Asp Leu Ala Lys Leu Ala Phe 145 150 155 160 Lys Lys Ser Ser Lys Tyr Asp Leu Glu Cys Ala Gln Ile Pro Val His 165 170 175 Met Arg Ser Asp Ala Ser Lys Tyr Thr His Glu Lys Pro Glu Gly His 180 185 190 Tyr Asn Trp His His Gly Ala Val Gln Tyr Ser Gly Gly Arg Phe Thr 195 200 205 Ile Pro Thr Gly Ala Gly Lys Pro Gly Asp Ser Gly Arg Pro Ile Phe 210 215 220 Asp Asn Lys Gly Arg Val Val Ala Ile Val Leu Gly Gly Ala Asn Glu 225 230 235 240 Gly Ser Arg Thr Ala Leu Ser Val Val Thr Trp Asn Lys Asp Met Val 245 250 255 Thr Arg Val Thr Pro Glu Gly Ser Glu Glu Trp Ser Ala Pro Leu Ile 260 265 270 Thr Ala Met Cys Val Leu Ala Asn Ala Thr Phe Pro Cys Phe Gln Pro 275 280 285 Pro Cys Val Pro Cys Cys Tyr Glu Asn Asn Ala Glu Ala Thr Leu Arg 290 295 300 Met Leu Glu Asp Asn Val Asp Arg Pro Gly Tyr Tyr Asp Leu Leu Gln 305 310 315 320 Ala Ala Leu Thr Cys Arg Asn Gly Thr Arg His Arg Arg Ser Val Ser 325 330 335 Gln His Phe Asn Val Tyr Lys Ala Thr Arg Pro Tyr Ile Ala Tyr Cys 340 345 350 Ala Asp Cys Gly Ala Gly His Ser Cys His Ser Pro Val Ala Ile Glu 355 360 365 Ala Val Arg Ser Glu Ala Thr Asp Gly Met Leu Lys Ile Gln Phe Ser 370 375 380 Ala Gln Ile Gly Ile Asp Lys Ser Asp Asn His Asp Tyr Thr Lys Ile 385 390 395 400 Arg Tyr Ala Asp Gly His Ala Ile Glu Asn Ala Val Arg Ser Ser Leu 405 410 415 Lys Val Ala Thr Ser Gly Asp Cys Phe Val His Gly Thr Met Gly His 420 425 430 Phe Ile Leu Ala Lys Cys Pro Pro Gly Glu Phe Leu Gln Val Ser Ile 435 440 445 Gln Asp Thr Arg Asn Ala Val Arg Ala Cys Arg Ile Gln Tyr His His 450 455 460 Asp Pro Gln Pro Val Gly Arg Glu Lys Phe Thr Ile Arg Pro His Tyr 465 470 475 480 Gly Lys Glu Ile Pro Cys Thr Thr Tyr Gln Gln Thr Thr Ala Lys Thr 485 490 495 Val Glu Glu Ile Asp Met His Met Pro Pro Asp Thr Pro Asp Arg Thr 500 505 510 Leu Leu Ser Gln Gln Ser Gly Asn Val Lys Ile Thr Val Gly Gly Lys 515 520 525 Lys Val Lys Tyr Asn Cys Thr Cys Gly Thr Gly Asn Val Gly Thr Thr 530 535 540 Asn Ser Asp Met Thr Ile Asn Thr Cys Leu Ile Glu Gln Cys His Val 545 550 555 560 Ser Val Thr Asp His Lys Lys Trp Gln Phe Asn Ser Pro Phe Val Pro 565 570 575 Arg Ala Asp Glu Pro Ala Arg Lys Gly Lys Val His Ile Pro Phe Pro 580 585 590 Leu Asp Asn Ile Thr Cys Arg Val Pro Met Ala Arg Glu Pro Thr Val 595 600 605 Ile His Gly Lys Arg Glu Val Thr Leu His Leu His Pro Asp His Pro 610 615 620 Thr Leu Phe Ser Tyr Arg Thr Leu Gly Glu Asp Pro Gln Tyr His Glu 625 630 635 640 Glu Trp Val Thr Ala Ala Val Glu Arg Thr Ile Pro Val Pro Val Asp 645 650 655 Gly Met Glu Tyr His Trp Gly Asn Asn Asp Pro Val Arg Leu Trp Ser 660 665 670 Gln Leu Thr Thr Glu Gly Lys Pro His Gly Trp Pro His Gln Ile Val 675 680 685 Gln Tyr Tyr Tyr Gly Leu Tyr Pro Ala Ala Thr Val Ser Ala Val Val 690 695 700 Gly Met Ser Leu Leu Ala Leu Ile Ser Ile Phe Ala Ser Cys Tyr Met 705 710 715 720 Leu Val Ala Ala Arg Ser Lys Cys Leu Thr Pro Tyr Ala Leu Thr Pro 725 730 735 Gly Ala Ala Val Pro Trp Thr Leu Gly Ile Leu Cys Cys Ala Pro Arg 740 745 750 Ala His Ala Ala Ser Val Ala Glu Thr Met Ala Tyr Leu Trp Asp Gln 755 760 765 Asn Gln Ala Leu Phe Trp Leu Glu Phe Ala Ala Pro Val Ala Cys Ile 770 775 780 Leu Ile Ile Thr Tyr Cys Leu Arg Asn Val Leu Cys Cys Cys Lys Ser 785 790 795 800 Leu Ser Phe Leu Val Leu Leu Ser Leu Gly Ala Thr Ala Arg Ala Tyr 805 810 815 Glu His Ser Thr Val Met Pro Asn Val Val Gly Phe Pro Tyr Lys Ala 820 825 830 His Ile Glu Arg Pro Gly Tyr Ser Pro Leu Thr Leu Gln Met Gln Val 835 840 845 Val Glu Thr Ser Leu Glu Pro Thr Leu Asn Leu Glu Tyr Ile Thr Cys 850 855 860 Glu Tyr Lys Thr Val Val Pro Ser Pro Tyr Val Lys Cys Cys Gly Ala 865 870 875 880 Ser Glu Cys Ser Thr Lys Glu Lys Pro Asp Tyr Gln Cys Lys Val Tyr 885 890 895 Thr Gly Val Tyr Pro Phe Met Trp Gly Gly Ala Tyr Cys Phe Cys Asp 900 905 910 Ser Glu Asn Thr Gln Leu Ser Glu Ala Tyr Val Asp Arg Ser Asp Val 915 920 925 Cys Arg His Asp His Ala Ser Ala Tyr Lys Ala His Thr Ala Ser Leu 930 935 940 Lys Ala Lys Val Arg Val Met Tyr Gly Asn Val Asn Gln Thr Val Asp 945 950 955 960 Val Tyr Val Asn Gly Asp His Ala Val Thr Ile Gly Gly Thr Gln Phe 965 970 975 Ile Phe Gly Pro Leu Ser Ser Ala Trp Thr Pro Phe Asp Asn Lys Ile 980 985 990 Val Val Tyr Lys Asp Glu Val Phe Asn Gln Asp Phe Pro Pro Tyr Gly 995 1000 1005 Ser Gly Gln Pro Gly Arg Phe Gly Asp Ile Gln Ser Arg Thr Val Glu 1010 1015 1020 Ser Asn Asp Leu Tyr Ala Asn Thr Ala Leu Lys Leu Ala Arg Pro Ser 1025 1030 1035 1040 Pro Gly Met Val His Val Pro Tyr Thr Gln Thr Pro Ser Gly Phe Lys 1045 1050 1055 Tyr Trp Leu Lys Glu Lys Gly Thr Ala Leu Asn Thr Lys Ala Pro Phe 1060 1065 1070 Gly Cys Gln Ile Lys Thr Asn Pro Val Arg Ala Met Asn Cys Ala Val 1075 1080 1085 Gly Asn Ile Pro Val Ser Met Asn Leu Pro Asp Ser Ala Phe Thr Arg 1090 1095 1100 Ile Val Glu Ala Pro Thr Ile Ile Asp Leu Thr Cys Thr Val Ala Thr 1105 1110 1115 1120 Cys Thr His Ser Ser Asp Phe Gly Gly Val Leu Thr Leu Thr Tyr Lys 1125 1130 1135 Thr Asn Lys Asn Gly Asp Cys Ser Val His Ser His Ser Asn Val Ala 1140 1145 1150 Thr Leu Gln Glu Ala Thr Ala Lys Val Lys Thr Ala Gly Lys Val Thr 1155 1160 1165 Leu His Phe Ser Thr Ala Ser Ala Ser Pro Ser Phe Val Val Ser Leu 1170 1175 1180 Cys Ser Ala Arg Ala Thr Cys Ser Ala Ser Cys Glu Pro Pro Lys Asp 1185 1190 1195 1200 His Ile Val Pro Tyr Ala Ala Ser His Ser Asn Val Val Phe Pro Asp 1205 1210 1215 Met Ser Gly Thr Ala Leu Ser Trp Val Gln Lys Ile Ser Gly Gly Leu 1220 1225 1230 Gly Ala Phe Ala Ile Gly Ala Ile Leu Val Leu Val Val Val Thr Cys 1235 1240 1245 Ile Gly Leu Arg Arg 1250 115 base pairs nucleic acid single linear RNA (genomic) NO NO - 1..115 /label= 26S_region /note= “26S promoter and transcription start and proximal downstream region of pSFV1; Figure 8.” misc_feature 1..24 /product= “26S promoter region” 4 ACCTCTACGG CGGTCCTAGA TTGGTGCGTT AATACACAGA ATCTGATTGG ATCCCGGGTA 60 ATTAATTGAA TTACATCCCT ACGCAAACGT TTTACGGCCG CCGGTGGCGC CCGCG 115 127 base pairs nucleic acid single linear RNA (genomic) NO NO - 1..127 /label= 26S_region /note= “26S promoter and transcription start and proximal downstream region of pSFV2; Figure 8.” misc_feature 1..24 /product= “26S promoter region” 5 ACCTCTACGG CGGTCCTAGA TTGGTGCGTT AATACACAGA ATTCTGATTA TAGCGCACTA 60 TTATATAGCA CCGGATCCCG GGTAATTAAT TGACGCAAAC GTTTTACGGC CGCCGGTGGC 120 GCCCGCG 127 123 base pairs nucleic acid single linear RNA (genomic) NO NO - 1..123 /label= 26S_region /note= “26S promoter and transcription start and proximal downstream region of pSFV3; Figure 8.” misc_feature 1..24 /product= “26S promoter region” 6 ACCTCTACGG CGGTCCTAGA TTGGTGCGTT AATACACAGA ATTCTGATTA TAGCGCACTA 60 TTATATAGCA CCATGGATCC CGGGTAATTA ATTGACGTTT TACGGCCGCC GGTGGCGCCC 120 GCG 123 54 base pairs nucleic acid single linear RNA (genomic) NO Semliki Forest Virus - 1..54 /label= restrict_site /note= “sequence of SFV E2 genome in vicinity of Bam HI site vector E2; Figure 12.” CDS 1..54 7 AAC TCA CCT TTC GTC CCG AGA GCC GAC GAA CCG GCT AGA AAA GGC AAA 48 Asn Ser Pro Phe Val Pro Arg Ala Asp Glu Pro Ala Arg Lys Gly Lys 1 5 10 15 GTC CAT 54 Val His 18 amino acids amino acid linear protein 8 Asn Ser Pro Phe Val Pro Arg Ala Asp Glu Pro Ala Arg Lys Gly Lys 1 5 10 15 Val His 46 base pairs nucleic acid double linear DNA (genomic) NO NO HIV - 1..46 /label= fragment /note= “HIV gp120 epitope introduced into SFV vector E2; Figure 12.” CDS 1..45 9 GAT CCG CGT ATC CAG AGA GGA CCA GGA AGA GCA TTT GTT GAG CTA 45 Asp Pro Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Glu Leu 1 5 10 15 G 46 15 amino acids amino acid linear protein 10 Asp Pro Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Glu Leu 1 5 10 15 51 base pairs nucleic acid double linear DNA (genomic) NO - 1..51 /label= chimaeric_seq /note= “SFV-HIV chimaeric sequence shown in Figure 12.” CDS 1..51 /product= “SFV-HIV chimaeric sequence” 11 GAG GAT CCG CGT ATC CAG AGA GGA CCA GGA AGA GCA TTT GTT GAG GAT 48 Glu Asp Pro Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Glu Asp 1 5 10 15 CCG 51 Pro 17 amino acids amino acid linear protein 12 Glu Asp Pro Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Glu Asp 1 5 10 15 Pro 60 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..60 /label= oligonucleotide /note= “used to introduce new linker site” 13 CGGCCAGTGA ATTCTGATTG GATCCCGGGT AATTAATTGA ATTACATCCC TACGCAAACG 60 62 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..62 /label= oligonucleotide /note= “used to introduce new linker site” 14 GCGCACTATT ATAGCACCGG CTCCCGGGTA ATTAATTGAC GCAAACGTTT TACGGCCGCC 60 GG 62 62 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..62 /label= oligonucleotide /note= “used to introduce new linker site” 15 GCGCACTATT ATAGCACCAT GGATCCGGGT AATTAATTGA CGTTTTACGG CCGCCGGTGG 60 CG 62 21 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..21 /label= primer /note= “SP1 upstream sequencing primer” 16 CGGCGGTCCT AGATTGGTGC G 21 21 base pairs nucleic acid single linear DNA (genomic) NO YES - 1..21 /label= primer /note= “SP2 downstream sequencing primer” 17 CGCGGGCGCC ACCGGCGGCC G 21 21 base pairs nucleic acid single linear DNA (genomic) NO YES - 1..21 /label= primer /note= “primer-1 for first strand cDNA synthesis” 18 TTTCTCGTAG TTCTCCTCGT C 21 27 base pairs nucleic acid single linear DNA (genomic) NO YES - 1..27 /label= primer /note= “primer-2 for first strand cDNA synthesis” 19 GTTATCCCAG TGGTTGTTCT CGTAATA 27 28 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..28 /label= primer /note= “5′ most primer for second strand cDNA synthesis, equals bp 1-28 of SFV sequence” 20 ATGGCGGATG TGTGACATAC ACGACGCC 28 46 base pairs nucleic acid double linear DNA (genomic) NO - 1..46 /label= adaptor /note= “5′-sticky end (EcoRI-HindIII-NotI-XmaIII-SpeI) blunt end-3′ adaptor” 21 AATTCAAGCT TGCGGCCGCA CTAGTGTTCG AACGCCGGCG TGATCA 46 8 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..8 /label= oligonucleotide /note= “NcoI oligonucleotide” 22 GCCATGGC 8 20 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..20 /label= oligonucleotide /note= “oligonucleotide used for screening by colony hybridization” 23 GGTGACACTA TAGCCATGGC 20 24 base pairs nucleic acid single linear DNA (genomic) NO NO - 1..24 /label= oligonucleotide /note= “site-directed mutagenic oligonucleotide used to introduce a BamHI site into the SFV genome” 24 GATCGGCCTA GGAGCCGAGA GCCC 24 80 base pairs nucleic acid single linear RNA (genomic) NO NO Semliki Forest Virus - 1..80 /label= terminator /note= “3′ terminal sequence of cDNA expression vector complementary to alphavirus genomic RNA” 25 TTTCCAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60 AAAAAAAAAA AAAAACTAGT 80 54 base pairs nucleic acid single linear RNA (genomic) NO Semliki Forest Virus - 1..54 /label= restrict_site /note= “sequence of SFV vector E2 in vicinity of Bam HI site; 12.” mutation 27..32 /label= restriction_sit /note= “BamHI recognition sequence introduced into SFV E2 genome in SFV vector E2.” CDS 1..54 26 AAC TCA CCT TTC GTC CCG AGA GCC GAG GAT CCG GCT AGA AAA GGC AAA 48 Asn Ser Pro Phe Val Pro Arg Ala Glu Asp Pro Ala Arg Lys Gly Lys 1 5 10 15 GTC CAT 54 Val His 18 amino acids amino acid linear protein 27 Asn Ser Pro Phe Val Pro Arg Ala Glu Asp Pro Ala Arg Lys Gly Lys 1 5 10 15 Val His 

1. An RNA molecule derived from an alphavirus RNA genome and capable of efficient infection of animal host cells, which RNA molecule comprises the complete alphavirus RNA genome regions, which are essential to replication of the said alpha-virus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof.
 2. The RNA of claim 1, wherein the said alphavirus is Semliki Forest virus (SFV).
 3. The RNA of claim 1 or 2, wherein the exogenous RNA sequence encodes a protein, a polypeptide or a peptide sequence defining an exogenous antigenic epitope or determinant.
 4. The RNA of claim 3 wherein the exogenous RNA sequence encodes an epitope sequence of a structural protein of an immunodeficiency virus inclusive of the human immunodeficiency virus (HIV) types.
 5. The RNA of any preceding claim, wherein the alphavirus derived RNA molecule regions comprise a 5′ terminal portion, the coding region(s) for non structural proteins required for RNA replication, the subgenome promoter region and a 3′ terminal portion of said viral RNA.
 6. The RNA of claim 2, 3 or 5, wherein the exogenous RNA sequence encodes a foreign polypeptide or protein and is integrated into the SFV subgenomic 26S RNA substituting deleted parts thereof.
 7. The RNA of claim 2, 3, 4 or 5, wherein the exogenous RNA sequence encodes a foreign viral epitopic peptide sequence and is located in a region of the RNA coding for structural alphavirus proteins enabling the exogenous RNA to be expressed as said viral epitope as part of the matured virus particle.
 8. The RNA of claim 2, 3, 4 or 5, wherein the exogenous RNA sequence encodes a foreign viral epitopic peptide sequence inserted into the p62 spike precursor subunit encoding region of the SFV genome.
 9. An RNA expression vector comprising the RNA of any preceding claim packaged into infectious particles comprising the RNA within the alphavirus nucleocapsid and surrounded by membrane with alphavirus spike proteins.
 10. The vector of claim 9, wherein the RNA has a total size corresponding to the wild type alphavirus RNA genome or deviating therefrom to an extent compatible with package of the RNA into the infectious particles.
 11. DNA transcription vector comprising a cDNA having one strand complementary to the RNA of any of claims 1 to
 8. 12. A DNA expression vector comprising a full-length or partial cDNA complementary to alphavirus RNA or parts thereof and located immediately downstream of the SP6 RNA polymerase promoter and having a 5′ATGG or 5′GATGG or any other 5′ terminus and a TTTCCA₆₉ACTAGT or any other 3′ terminus.
 13. The vector of claim 12 having portions of the viral cDNA deleted, the deletions comprising the complete or part of the region(s) encoding the virus structural proteins, and further comprising an integrated polylinker region, which may correspond to BamHI-SmaI-XmaI, inserted at a location which enables an exogenous DNA fragment encoding a foreign polypeptide or protein to be inserted into the vector cDNA for subsequent expression in an animal host cell.
 14. The vector of claim 12 or 13 wherein the alphavirus is SFV.
 15. The vector of claim 12 or 14 comprising full-length cDNA and further comprising an exogenous DNA fragment encoding a foreign epitopic peptide sequence or antigenic determinant inserted into a region of the viral structural proteins.
 16. The vector of claim 15 wherein the exogenous DNA fragment is inserted into the p62 spike precursor subunit encoding region of the SFV cDNA.
 17. The vector of any preceding claim comprising an SFV derived cDNA which carries a conditionally lethal SFV mutation in the region encoding the p62 cleavage site, a cellularly uncleavable but extracellularly cleavable form of p62 being expressed.
 18. The vector of claim 13 comprising SFV-derived cDNA, the vector being pSFV1, pSFV2 or pSFV3 having a structure as shown in FIG.
 8. 19. An RNA transcript derived from transcription of the DNA-vector of any of claims 12-18 carrying an exogenous DNA fragment.
 20. A method to produce the vector of claim 9 or 10 wherein the alphavirus derived RNA lacks part of or the complete region(s) encoding the structural viral proteins, the method comprising cotransfection of animal host cells with the RNA transcript of claim 19, wherein the alphavirus RNA lacks part(s) of or the complete region(s) encoding the viral structural proteins, with helper RNA transcribed in vitro from a helper DNA vector and culturing the host cells.
 21. The method of claim 20 wherein the cotransfection is produced by electroporation of the host cells.
 22. Helper vector for use in the method according to claim 20 or 21, said vector being comprised of the DNA vector of claim 12 wherein the regions encoding non structural virus proteins are almost completely deleted, including sequences encoding RNA signals for packaging of RNA into nucleocapsid particles, but the 5′ and 31 signals needed for RNA replication and the region encoding the promoter for the structural sub-genome are in addition to those encoding the structural region preserved.
 23. Helper vector of claim 22 wherein the cDNA has its origin from SFV and the deletion extends from the AccI (308) to the AccI (6399) restriction endonuclease site of the full-length cDNA vector of claim
 12. 24. Helper vector of claims 22 and 23 where the structural region contains the mutation described in claim 17 or another conditionally lethal mutation.
 25. The method of claim 20 wherein cells transformed to produce helper RNA according to claims 20, 22 or 23 are transfected with RNA transcript of claim
 19. 26. A host cell of animal origin transformed with the RNA of any of claims 1-8, the DNA transcription vector of claims 11 or the DNA vector of any of claims 12-18 carrying an exogenous DNA fragment.
 27. The host cell of claim 26 wherein the cell is an avian, a mammalian, a reptilian, an amphibian, an insecticidal or a fish cell.
 28. The host cell of claim 27 which is the hamster BHK cell.
 29. A method to produce the transformed host cell of claim 26, 27 or 28 comprising transfection of the cell with the RNA of any of claims 1-8, with the cDNA of claim 11 or of any of claims 12-18 carrying an exogenous DNA fragment or infection of the cell with the infectious viral particles of claim 9 or
 10. 30. The method of claim 29 wherein the transfection is produced by electroporation of the host cell.
 31. A method for the production of a polypeptide or protein comprising infection of animal host cells with infectious particles according to claim 9 or 10, containing exogenous RNA encoding said polypeptide or protein and produced according to method of claim 20 or 21, culturing the said transformed cells to express the exogenous RNA and isolation and purification of the product formed by said expression.
 32. A method for the production of a polypeptide or protein comprising in vitro transcription of the cDNA of the vector of any of claims 11-18 carrying an exogenous DNA fragment coding for the polypeptide or protein, transfection of animal host cells with the produced RNA transcript, transformed animal host cells being obtained harbouring the RNA transcript, culturing the said transformed cells to express the exogenous RNA and isolation and purification of the product formed by said expression.
 33. The metod of claim 32 wherein the vector cDNA is comprised of the cDNA of the vector of claim 17 carrying the exogenous DNA fragment.
 34. An antigen consisting of a chimaeric alphavirus having an exogenous epitopic peptide sequence or antigenic determinant inserted into its structural proteins.
 35. The antigen of claim 34 wherein the chimaeric alpha-virus is derived from SFV.
 36. The antigen of claim 34 or 35, wherein the exogenous epitopic peptide sequence is comprised of an epitopic peptide sequence derived from a structural protein of a virus belonging to the immunodeficiency virus class inclusive of the human immunodeficiency virus types.
 37. Vaccine preparation comprising the antigen of claim 34, 35 or 36 as immunizing component.
 38. Vaccine of claim 37 wherein the chimaeric alphavirus is attenuated by comprising the conditionally lethal SFV mutation of claim 17, an amber (stop codon) a temperature sensitive mutation or other mutation in its genome.
 39. A method for the production of an antigen of claim 34, 35 or 36 comprising a) in vitro transcription of the cDNA of the vector of any of claims 11-18 carrying an exogenous DNA fragment encoding the foreign epitopic peptide sequence or antigenic determinant and transfection of animal host cells with the produced RNA transcript, or b) transfection of animal host cells with the said cDNA of the above step a), culturing the transfected cells and recovering the chimaeric alphavirus antigen.
 40. The method of claim 32, 33 or 39 wherein the transfection is produced by electroporation of the host cell.
 41. A method for the production of an antigen in an organism by using in vivo infection with infectious particles according to claim 9 or 10 containing exogenous RNA encoding an exogenous epitopic peptide sequence or antigenic determinant, and produced according the claim 20 or
 21. 